US 2011 0191902A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0191902 A1 Atkinson et al. (43) Pub. Date: Aug. 4, 2011
(54) METHODS AND COMPOSITIONS FOR Publication Classification INCREASING STORAGE-LIFE OF FRUIT (51) Int. Cl. AOIH I/00 (2006.01) (76) Inventors: Ross Graham Atkinson, Auckland CI2N 5/10 (2006.01) (NZ); Robert James Schaffer, AOIH 5/00 (2006.01) Auckland (NZ); Kularajathevan CI2N 5/82 (2006.01) Gunaseelan, Auckland (NZ); C7H 2L/00 (2006.01) Roswitha Schröder, Auckland CI2N 9/14 (2006.01) (NZ) (52) U.S. Cl...... 800/278; 435/419: 800/298; 435/320.1; 800/301; 536/23.2:435/195 (21) Appl. No.: 13/060,257 (57) ABSTRACT The invention provides methods and compositions for pro (22) PCT Filed: Aug. 28, 2009 ducing plants with fruit having increased post-harvest storage life, the method comprising reducing the expression or activ (86). PCT No.: PCT/NZ09/00182 ity in the plant, of a polypeptide with the amino acid sequence of SEQID NO: 1, or a variant of the polypeptide. The inven S371 (c)(1), tion provides host cells, plant cells and plants transformed (2), (4) Date: Apr. 14, 2011 with the polynucleotides of the invention. The invention also provides methods for selecting plants with fruit having (30) Foreign Application Priority Data increased post-harvest storage life. The invention also pro vides plants produced and selected by the methods of the Aug. 29, 2008 (NZ) ...... 570886 invention.
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METHODS AND COMPOSITIONS FOR radation in fruit. However, no significant effect on fruit soft INCREASING STORAGE-LIFE OF FRUIT ening, ethylene evolution, or color development was detected. The authors reported that “polygalacturonase was TECHNICAL FIELD the primary determinant of cell wall polyuronide degradation, but suggested that this degradation was not sufficient for the 0001. The present invention relates methods and compo induction of softening (Giovannoni et al., 1989). sitions for producing plants with fruit with increased storage 0010 Further experiments where the pTOM6 gene was life. overexpressed in tobacco (Nicotiana tabacum; Osteryoung et al., 1990) showed that the tomato protein was properly pro BACKGROUND ART cessed and localized in the cell walls of leaves in tobacco. The 0002 Post-harvest spoilage is a major problem for the enzyme showed activity when extracted from transgenic fruit industry. It has been estimated that 10-20% of post tobacco leaves and tested against tobacco cell wall extracts in harvest fruit is lost through spoilage before reaching consum vitro. However, no changes in leaf phenotype were observed, ers. This spoilage results in increased cost of the non-spoiled nor were there any alterations to the pectins in the tobacco cell fruit to the consumer. In addition fruit is often discarded by walls in vivo. the consumer because of spoilage after purchase but before 0011 Together these results suggested to researchers that the fruit is eaten. PG only had role in pectin depolymerization primarily in fruit 0003. One of the main causes of spoilage is the natural but the enzyme did not have an affect on fruit softening. ripening of fruit. As fruit ripens it tends to become softer and 0012. Thus in spite of such substantial research the prob more Susceptible to mechanical damage, as well as biological lem of post-harvest softening has not been overcome for most damage from necrophytic pathogens Such as storage rots. fruits and particularly in apple. Apple (Malus domestica 0004. In addition to the problems associated with soften Borkh.cv Royal Gala) ripens very differently than tomato and ing, the flesh of fruits such as apples often develop a “mealy' many other fruits because cell wall swelling is not one of the dry texture during post-harvest storage, which is unpopular cell wall modifications occurring during apple ripening with consumers. Mealy texture is believed to be a result of (Redgwell et al., 1997). There is minimal change in viscosity separation of cells in the flesh when the fruit is bitten, without of cell walls, and minimal pectin Solubilization or degrada associated rupture of cells and release of the apple's juice. tion during fruit ripening. It would therefore be of benefit to This creates the impression of a dry, less juicy apple, as well provide improved or alternative methods to addressing post as a less crunchy or crispy apple. harvest Softening in apples and other fruit. 0005 Water loss from fruit during storage is also a prob 0013. It is an object of the invention to provide improved lem and can lead to fruit developing an unattractive shriveled methods and compositions for producing plants with fruit appearance. having increased post-harvest storage life, or at least to pro 0006 Various approaches have been used to attempt to vide the public with a useful choice. address post-harvest deterioration of fruit. For example, genetic approaches have focused on manipulating the expres SUMMARY OF THE INVENTION sion of ethylene biosynthesic genes such as ACC oxidase (e.g. 0014. In a first aspect the invention provides a method for in apple, Schafferetal 2007; tomato, Hamilton etal 1990; and producing a plant with fruit having increased post-harvest melon, Ayub etal 1996), and ACC synthase (Oelleretal 1991) storage life, the method comprising reducing the expression and on genes that influence cell wall physiology Such as or activity in the plant, of a polypeptide with the amino acid pectin lyase (Santiago-Doménech et al 2008), expansin sequence of SEQID NO: 1, or a variant of the polypeptide. (Brummelletal 1999) and B-galactosidase (Carey etal 2001). 0015. In one embodiment the fruit have at least one of: However, to the applicants knowledge no fruits resulting from 0016 a) increased firmness, Such approaches are currently commercially available. (0017 b) reduced water loss, 0007 Transgenic tomatoes in which expression of a 0018 c) reduced cell separation, polygalacturonase (PG) gene was reduced were commercia 0019 d) increased juiciness, lised as the well documented Flavir Savr tomatoes. However 0020 e) increased crispiness, technical problems reportedly made it difficult to ship the 0021 f) increased waxiness, and delicate GE tomatoes without damage. Sale of Flavir Savr 0022 g) reduced Susceptibility to necrophytic pathogens, tomatoes was ultimately withdrawn. during, or after, post-harvest storage. 0008. From a scientific perspective tomato fruit with (0023 Preferably the fruit have at least two, more prefer altered levels of polygalacturonase have been studied exten ably at least three, more preferably at least four, more pref sively. Tomato fruit containing an antisense PG gene erably at least five, more preferably at least six, most prefer (pTOM6) showed reduced depolymerization of pectin poly ably all, of a) to g). mers in fruit (Smith et al., 1990). However “the firmness of 0024. In a preferred embodiment the fruit have increased the fruit throughout ripening was not altered in the transgenic firmness. samples when compared to controls” (Schuch et al 1991). 0025 Preferably in addition to increased firmness, the Postharvest cracking, rates of infection, and the ability to fruit also show at least one ofb) to g). Preferably, in addition withstand transport were improved in the antisense PG toma to increased firmness, the fruit preferably have at least two, toes. Many other studies on these transgenic lines Support the more preferably at least three, more preferably at least four, role for PG in pectin depolymerization but not fruit softening more preferably at least five, and most preferably all, ofb) to (Carrington et al 1993, Cantu et al 2008: Langley et al 1994, g). Powell et al 1993) 0026. In a further embodiment the variant comprises a 0009. Overexpression of PG in the ripening inhibited sequence with at least 70% identity to the amino acid mutant rin background restored PG activity and pectin deg sequence of SEQID NO: 1. US 2011/O 191902 A1 Aug. 4, 2011
0027. In a further embodiment the variant comprises the 0048. In a further embodiment the polynucleotide and a sequence of SEQID NO: 2. sequence complimentary to the polynucleotide are included 0028. In a further embodiment the variant comprises the in the RNAi construct to form a double stranded RNA when sequence of SEQID NO:3. the polynucleotide and sequence complimentary thereto are 0029. In a further embodiment the variant has polygalac transcribed. turonase activity. 0049. In a further embodiment the polynucleotide com prises at least 15 contiguous nucleotides of a sequence with at Reducing Expression of the Polypeptide by Introducing a least 70% identity to the sequence of SEQID NO: 4. Polynucleotide 0050. In a further embodiment the polynucleotide com prises at least 15 contiguous nucleotides of the sequence of 0030. In a further embodiment the method comprises the SEQID NO: 4. step of introducing a polynucleotide into a plant cell, or plant, 0051. In a further embodiment the polynucleotide com to effect reducing the expression of the polypeptide or variant. prises at least 15 contiguous nucleotides of a sequence with at least 70% identity to the sequence of SEQID NO: 5. Targeting Gene Encoding Polypeptide Using Complemen 0052. In a further embodiment the polynucleotide com tary Polynucleotide prises at least 15 contiguous nucleotides of the sequence of 0031. In a further embodiment the polynucleotide com SEQID NO: 5. prises a sequence with at least 70% identity to part of an 0053. In a further embodiment the polynucleotide com endogenous gene, or nucleic acid, that encodes the polypep prises at least 15 contiguous nucleotides of the sequence of tide or variant thereof. SEQID NO: 6. 0032. In a further embodiment the polynucleotide com 0054. In a further embodiment the polynucleotide com prises a sequence that hybridises understringent conditions to prises at least 15 contiguous nucleotides of the sequence of part of an endogenous gene, or nucleic acid, encoding the SEQID NO: 7. polypeptide, or a variant of the polypeptide. 0055. In a further embodiment a plant with reduced 0033. The part of the endogenous gene may include part of expression of the polypeptide is regenerated from the plant an element selected from the promoter, a 5' untranslated cell. sequence (UTR), an exon, an intron, a 3' UTR or the termi 0056. In a further aspect the invention provides a plant nator of the gene, or a may span more than one of Such produced by the method. elements. 0034. In a further embodiment the endogenous gene has at Silencing Constructs and Cells and Plants Containing the least 70% identity to the sequence of SEQID NO: 4. Silencing Constructs 0035. In a further embodiment the endogenous gene has 0057. In a further aspect the invention provides an expres the sequence of SEQID NO: 4. sion construct comprising a promoter operably linked to a 0036. In a further embodiment the endogenous nucleic polynucleotide comprising a fragment of at least 15 contigu acid comprises a sequence with at least 70% identity to the ous nucleotides of a sequence with at least 70% identity to any sequence of SEQID NO: 5. one of SEQID NO: 4, 5, 6 and 7, wherein the sequence with 0037. In a further embodiment the endogenous nucleic 70% identity to any one of SEQID NO: 4, 5, 6 and 7, encodes acid comprises the sequence of SEQID NO: 5. a polypeptide with polygalacturonase activity. 0038. In a further embodiment the endogenous nucleic 0058. In one embodiment the polynucleotide comprises a acid comprises the sequence of SEQID NO: 6. fragment of at least 15 contiguous nucleotides any one of 0039. In a further embodiment the endogenous nucleic SEQID NO: 4, 5, 6 and 7. acid comprises the sequence of SEQID NO: 7. 0059. In a further embodiment the polynucleotide in an antisense orientation relative to the promoter. 0040. In a further embodiment the polynucleotide com 0060. In a further embodiment the expression construct is prises at least 15 contiguous nucleotides that are at least 70% an RNAi construct. identical to part of the endogenous gene or nucleic acid. 0061. In a further embodiment the polynucleotide and a 0041. In a further embodiment the polynucleotide com sequence complimentary to the polynucleotide are included prises at least 15 contiguous nucleotides that hybridise under in the RNAi construct to form the hairpin loop of the RNAi stringent conditions to the endogenous gene or nucleic acid. COnStruct. 0042. In a further embodiment the polynucleotide is intro 0062. In a further embodiment the polynucleotide and a duced into the plant as part of a genetic construct. sequence complimentary to the polynucleotide are included 0043. In a further embodiment the genetic construct is an in the RNAi construct to form a double stranded RNA when expression construct comprising a promoter operably linked the polynucleotide and sequence complimentary thereto are to the polynucleotide. transcribed. 0044. In a further embodiment the polynucleotide in a 0063. In a further embodiment the invention provides a sense orientation relative to the promoter. plant cell, or plant, comprising an expression construct of the 0045. In a further embodiment the polynucleotide in an invention. antisense orientation relative to the promoter. 0064 Preferably the plant cell of plant has modified 0046. In a further embodiment the expression construct is expression of the endogenous nucleic acid corresponding to an RNAi construct. the polynucleotide. 0047. In a further embodiment the polynucleotide and a 0065 Preferably the endogenous nucleic acid encodes a sequence complimentary to the polynucleotide are included polypeptide with polygalacturonase activity. in the RNAi construct to form the hairpin loop of the RNAi 0066. In further embodiment the planthas, or is capable of COnStruct. producing, fruit with increased post-harvest storage life. US 2011/O 191902 A1 Aug. 4, 2011
0067. In one embodiment the fruit have at least one of: 0093. In a further aspect the invention provides an isolated 0068 a) increased firmness, polypeptide comprising the amino acid sequence of SEQID 0069 b) reduced water loss, NO: 2 or 3, or a variant thereof, wherein the variant has 0070 c) reduced cell separation, polygalacturonase activity. 0071 d) increased juiciness, 0094. In one embodiment the variant polypeptide has at 0072 e) increased crispiness, least 90% sequence identity to an amino acid sequence of 0073 f) increased waxiness, and SEQID NO: 2 or 3. 007.4 g) reduced susceptibility to necrophytic pathogens, 0095. In a further embodiment the isolated polypeptide during, or after, post-harvest storage. has at least 90% sequence identity to the amino acid sequence 0075 Preferably the fruit have at least two, more prefer of SEQID NO: 2. ably at least three, more preferably at least four, more pref 0096. In a further embodiment the isolated polypeptide erably at least five, more preferably at least six, most prefer comprises the amino acid sequence of SEQID NO: 2. ably all, of a) to g). 0097. In a further embodiment the isolated polypeptide 0076. In a preferred embodiment the fruit have increased has at least 90% sequence identity to the amino acid sequence firmness. of SEQ ID NO:3. 0077 Preferably in addition to increased firmness, the 0098. In a further embodiment the isolated polypeptide fruit also show at least one ofb) to g). Preferably, in addition comprises the amino acid sequence of SEQID NO: 3. to increased firmness, the fruit preferably have at least two, 0099. In a further aspect the invention provides an isolated more preferably at least three, more preferably at least four, polynucleotide encoding a polypeptide of the invention. more preferably at least five, and most preferably all, ofb) to 0100. In a further aspect the invention provides an isolated g). polynucleotide comprising: 0078. In a further aspect the invention provides a method 0101 a) a polynucleotide comprising a fragment, of at for selecting a plant with, or capable of producing, fruit hav ing increased post-harvest storage life, the method compris least 15 nucleotides in length, of a polynucleotide of the ing testing of a plant for altered expression of a polynucle invention; otide encoding a polypeptide with at least 70% identity to the 0102 b) a polynucleotide comprising a complement, of sequence of SEQID NO: 1. at least 15 nucleotides in length, of the polynucleotide of 0079. In a further aspect the invention provides a method the invention; or for selecting a plant with, or capable of producing, fruit hav 0.103 d) a polynucleotide comprising a sequence, of at ing increased post-harvest storage life, the method compris least 15 nucleotides in length, capable of hybridising to ing testing of a plant for altered expression of a polypeptide the polynucleotide of the invention. with at least 70% identity to the sequence of SEQID NO: 1. 0104. In a further aspect the invention provides a genetic 0080. In one embodiment of the above two aspects, the construct which comprises a polynucleotide of the invention. polypeptide has the sequence of SEQID NO: 2. 0105. In a further aspect the invention provides an expres 0081. In a further embodiment of the above two aspects, sion construct which comprises a polynucleotide of the inven the polypeptide has the sequence of SEQID NO: 3. tion. 0082 In a further aspect the invention provides a group or 0106. In a further aspect the invention provides an RNAi population of plants selected by the method of the invention. construct which comprises a polynucleotide of the invention. 0083. In a further aspect the invention provides an isolated 0107. In a further aspect the invention provides a vector polynucleotide encoding a polypeptide comprising a comprising an expression construct, genetic construct or sequence of SEQID NO: 2 or 3 or a variant thereof wherein RNAi construct of the invention. the variant has polygalacturonase activity. 0108. In a further aspect the invention provides a host cell 0084. In one embodiment the variant comprises a genetically modified to express a polynucleotide of the inven sequence with at least 90% identity to SEQID NO: 2: tion, or a polypeptide of the invention. 0085. In a further embodiment the polypeptide comprises 0109. In a further aspect the invention provides a host cell the sequence of SEQID NO: 2. comprising an expression construct or genetic construct of I0086. In one embodiment the variant comprises a the invention. sequence with at least 90% identity to SEQID NO:3: 0110 Preferably the host cell is a plant cell. Preferably the 0087. In a further embodiment the polypeptide comprises plant cell is part of a plant. the sequence of SEQID NO:3. 0111. In a further aspect the invention provides a plant cell 0088. In a further aspect the invention provides an isolated genetically modified to express a polynucleotide of the inven polynucleotide comprising the sequence of SEQID NO: 6 or tion, or a polypeptide of the invention. 7, or a variant thereof wherein the variant encodes a polypep 0112. In a further aspect the invention provides a plant cell tide with polygalacturonase activity. which comprises an expression construct of the invention or 0089. In one embodiment the variant comprises a the genetic construct of the invention. sequence with at least 70% sequence identity to the sequence 0113. In a further aspect the invention provides a plant of SEQID NO: 6. which comprises a plant cell of the invention. 0090. In one embodiment the polynucleotide comprises 0114. The polynucleotides and variants of polynucle the sequence of any one of SEQID NO: 6. otides, of the invention, or used in the methods of the inven 0091. In one embodiment the variant comprises a tion, may be derived from any species. The polynucleotides sequence with at least 70% sequence identity to the sequence and variants may also be recombinantly produced and also of SEQ ID NO: 7. may be the products of "gene shuffling approaches. 0092. In one embodiment the polynucleotide comprises 0.115. In one embodiment the polynucleotide or variant, is the sequence of any one of SEQID NO: 7. derived from a plant species. US 2011/O 191902 A1 Aug. 4, 2011
0116. In a further embodiment the polynucleotide or vari Prunus fordiana, Prunus fremontii, Prunus fruticosa, Prunus ant, is derived from a gymnosperm plant species. geniculata, Prunus glandulosa, Prunus gracilis, Prunus 0117. In a further embodiment the polynucleotide or vari gravana, Prunus hortulana, Prunus ilicifolia, Prunus incisa, ant, is derived from an angiosperm plant species. Prunus jacquemontii, Prunus japonica, Prunus kuramica, 0118. In a further embodiment the polynucleotide or vari Prunus laurocerasus, Prunus leveilleana, Prunus lusitanica, ant, is derived from a from dicotyledonous plant species. Prunus maackii, Prunus mahaleb, Prunus mandshurica, Pru 0119 The polypeptides and variants of polypeptides of the nus maritima, Prunus maximowiczii, Prunus mexicana, Pru invention, or used in the methods of the invention, may be nus microcarpa, Prunus mira, Prunus mume, Prunus munso derived from any species. The polypeptides and variants may niana, Prunus nigra, Prunus nipponica, Prunus padus, also be recombinantly produced and also may also be Prunus pensylvanica, Prunus persica, Prunus petunnikowii, expressed from the products of 'gene shuffling approaches. Prunus prostrata, Prunus pseudocerasus, Prunus pumila, 0120 In one embodiment the polypeptides or variants of Prunus rivularis, Prunus Salicina, Prunus Sargentii, Prunus the invention are derived from plant species. sellowii, Prunus serotina, Prunus Serrulata, Prunus Sibirica, 0121. In a further embodiment the polypeptides or vari Prunus Simonii, Prunus spinosa, Prunus spinulosa, Prunus ants of the invention are derived from gymnosperm plant subcordata, Prunus Subhirtella, Prunus takesimensis, Pru species. nus tenella, Prunus texana, Prunus tomentosa, Prunus 0122. In a further embodiment the polypeptides or vari tSchonoski, Prunus umbellata, Prunus verecunda, Prunus ants of the invention are derived from angiosperm plant spe virginiana, Prunus webbii, PrunusXyedoensis, Prunus Zippe C1GS. liana, Prunus sp. BSP-2004-1, Prunus sp. BSP-2004-2, Pru 0123. In a further embodiment the polypeptides or vari nus sp. EB-2002, Amelanchier alnifolia, Amelanchier ants of the invention are derived from dicotyledonous plant arborea, Amelanchier asiatica, Amelanchier bartramiana, species. Amelanchier Canadensis, Amelanchier clusickii, Amelanchier 0.124. The plant cells and plants of the invention, including fernaldii, Amelanchier florida, Amelanchier humilis, those from which the polynucleotides, variant polynucle Amelanchier intermedia, Amelanchier laevis, Amelanchier otides, polypeptide and variant polypeptides are derived, may lucida, Amelanchier mantucketensis, Amelanchier pumila, be from any fruit species. Amelanchier quinti-martii, Amelanchier sanguinea, Amelan 0.125. In one embodiment the fruit are from Rosaceae spe chier stolonifera, Amelanchier utahensis, Amelanchier wie cies. gandii, Amelanchierxneglecta, Amelanchier bartramianax 0126 Preferred Rosaceae genera include Exochorda, Amelanchier sp. dentata, Amelanchier sp. dentata, Maddenia, Oemleria, Osmaronia, Prinsepia, Prunus, Mal Amelanchier sp. erecta, Amelanchier sp. erectaxAmelan Oideae, Amelanchier, Aria, Aronia, Chaenomeles, Chamae chier laevis, Amelanchier sp. serotina, Aria alnifolia, Aro mespilus, Cornus, Cotoneaster, Crataegus Osmaronia, Prin nia prunifolia, Chaenomeles cathayensis, Chaenomeles spe sepia, Prunus, Maloideae, Amelanchier, Aria, Aronia, ciosa, Chamaeme spilus alpina, Cornus domestica, Chaenomeles, Chamaemespilus, Cornus, Cotoneaster, Cra Cotoneaster apiculatus, Cotoneaster lacteus, Cotoneaster taegu, Cydonia, Dichotomanthes, Docynia, Docyniopsis, pannosus, Crataegus azarolus, Crataegus columbiana, Cra Eriobotrya, Eriolobus, Heteromeles, Kagemeckia, Lindleya, taegus crus-galli, Crataegus curvisepala, Crataegus laevi Malacomeles, Malus, Mespilus, Osteomeles, Peraphyllum, gata, Crataegus mollis, Crataegus monogyna, Crataegus Photinia, Pseudocydonia, Pyracantha, Pyrus, Rhaphiolepis, nigra, Crataegus rivularis, Crataegus Sinaica, Cydonia Sorbus, Stranvaesia, Torminalis, Vauquelinia, Rosoideae, oblonga, Dichotomanthes tristanicarpa, Docynia delavavi, Acaena, Acomastylis, Agrimonia, Alchemilla, Aphanes, Docyniopsis tschonoski, Eriobotrya japonica, Eriobotrya Aremonia, Bencomia, Chamaebatia, Cliffortia, Coluria, prinoides, Eriolobus trilobatus, Heteromeles arbutifolia, Cowania, Dalibarda, Dendriopoterium, Dryas, Duchesnea, Kagemeckia angustifolia, Kagemeckia Oblonga, Lindleya Erythrocoma, Fallugia, Filipendula, Fragaria, Geum, Hage mespiloides, Malacomeles denticulata, Malus angustifolia, nia, Horkelia, Ivesia, Kerria, Leucosidea, Marcetella, Mar Malus asiatica, Malus baccata, Malus coronaria, Malus gyricarpus, Novosieversia, Oncostylus, Polylepis, Potentilla, doumeri, Malus florentina, Malus floribunda, Malus fisca, Rosa, Rubus, Sanguisorba, Sarcopoterium, Sibbaldia, Siev Malus halliana, Malus homanensis, Malus hupehensis, Malus ersia, Taihangia, Tetraglochin, Waldsteinia, Rosaceae incer ioensis, Malus kansuensis, Malus mandshurica, Malus tae sedis, Adenostoma, Aruncus, Cercocarpus, Chamaeba micromalus, Malus niedzwetzkyana, Malus Ombrophilia, tiaria, Chamaerhodos, Gillenia, Holodiscus, Lyonothamnus, Malus Orientalis, Malus pratti, Malus prunifolia, Malus Neillia, Neviusia, Physocarpus, Purshia, Rhodotypos, Sor punila, Malus Sargentii, Malus sieboldii, Malus sieversii, baria, Spiraea and Stephanandra. Malus Sylvestris, Malus toringoides, Malus transitoria, 0127 Preferred Rosaceae species include: Exochorda Malus trilobata, Malus tschonoskii, MalusXdomestica, giraldi, Exochorda racemosa, Exochorda, Exochorda giral MalusXdomesticaxMalus sieversii, MalusXdomesticaXPyrus dii, Exochorda racemosa, Exochorda Serratifolia, Maddenia communis, Malus xiaojinensis, Malus yunnanensis, Malus hypoleuca, Oemleria cerasiformis, Osmaronia cerasiformis, sp., Mespilus germanica, Osteomeles anthyllidifolia, Prinsepia sinensis, Prinsepia uniflora, Prunus alleghanien Osteomeles Schwerinae, Peraphyllum ramosissimum, Pho sis, Prunus americana, Prunus andersonii, Prunus angusti tinia fraseri, Photinia pyrifolia, Photinia serrulata, Photinia folia, Prunus apetala, Prunus argentea, Prunus armeniaca, villosa, Pseudocydonia sinensis, Pyracantha coccinea, Pyra Prunus avium, Prunus bifrons, Prunus brigantina, Prunus cantha fortuneana, Pyrus calleryana, Pyrus caucasica, bucharica, Prunus buergeriana, Prunus campanulata, Pru Pyrus communis, Pyrus elaeagrifolia, Pyrus hybrid cultivar, nus Caroliniana, Prunus cerasifera, Prunus cerasus, Prunus Pyrus pyrifolia, Pyrus salicifolia, Pyrus ussuriensis, PyrusX choreiana, Prunus coconilia, Prunus cyclaimina, Prunus bretschneideri, Rhaphiolepis indica, Sorbus americana, Sor davidiana, Prunus debilis, Prunus domestica, Prunus dulcis, bus aria, Sorbus aucuparia, Sorbus Californica, Sorbus com Prunusemarginata, Prunus fasciculata, Prunus ferganensis, mixta, Sorbus hupehensis, Sorbus scopulina, Sorbus Sibirica, US 2011/O 191902 A1 Aug. 4, 2011
Sorbus torminalis, Stranvaesia davidiana, Torminalis clusii, tida, Rosa foliolosa, Rosa gallica, Rosa gallicaxRosa dune Vauquelinia Californica, Vauquelinia corymbosa, Acaena torum, Rosa gigantea, Rosa glauca, Rosa helenae, Rosa hen anserinifolia, Acaena argentea, Acaena Caesiglauca, rvi, Rosa hugonis, Rosa hybrid cultivars Rosa inodora, Rosa Acaena cylindristachya, Acaena digitata, Acaena echinata, jundzillii, Rosa laevigata, Rosa taxa, Rosa luciae, Rosa maja Acaena elongata, Acaena eupatoria, Acaena fissistipula, lis, Rosa marretii, Rosa maximowicziana, Rosa micrantha, Acaena inermis, Acaena laevigata, Acaena latebrosa, Rosa mollis, Rosa montana, Rosa moschata, Rosa movesii, Acaena lucida, Acaena macrocephala, Acaena magellanica, Rosa multibracteata, Rosa multiflora, Rosa initida, Rosa Odo Acaena masafuerana, Acaena montana, Acaena multifida, rata, Rosa palustris, Rosa pendulina, Rosa persica, Rosa Acaena novaezelandiae, Acaena ovalifolia, Acaena pinnati phoenicia, Rosa platyacantha, Rosa primula, Rosa pseudos fida, Acaena splendens, Acaena subincisa, Acaenaxanse cabriuscula, Rosa roxburghii, Rosa rubiginosa, Rosa rugosa, rovina, Acomastylis elata, Acomastylis rossii, Acomastylis Rosa Sambucina, Rosa sempervirens, Rosa sericea, Rosa ser Sikkimensis, Agrimonia eupatoria, Agrimonia nipponica, tata, Rosa setigera, Rosa Sherardii, Rosa Sicula, Rosa spino Agrimonia parviflora, Agrimonia pilosa, Alchemilla alpina, sissima, Rosa stellata, Rosa stylosa, Rosa subcanina, Rosa Alchemilla erythropoda, Alchemilla japonica, Alchemilla subcollina, Rosa sufiulta, Rosa tomentella, Rosa tomentosa, mollis, Alchemilla vulgaris, Aphanes arvensis, Aremonia Rosa tunquinensis, Rosa villosa, Rosa virginiana, Rosa agrimonioides, Bencomia brachystachya, Bencomia cau wichurana, Rosa willmottiae, Rosa woodsii, Rosaxdama data, Bencomia existipulata, Bencomia sphaerocarpa, scena, Rosaxfortuniana, Rosaxmacrantha, Rosa xanthina, Chamaebatia foliolosa, Cliffortia burmeana, Cliffortia Rosa sp., Rubus alceifolius, Rubus allegheniensis, Rubus cuneata, Cliffortia dentata, Cliffortia graminea, Cliffortia alpinus, Rubus amphidasys, Rubus arcticus, Rubus argutus, heterophylla, Cliffortianitidula, Cliffortia odorata, Cliffortia Rubus assamensis, Rubus australis, Rubus bifrons, Rubus ruscifolia, Cliffortia sericea, Coluria elegans, Coluria caesius, Rubus caesiusXRubus idaeus, Rubus Canadensis, geoides, Cowania Stansburiana, Dalibarda repens, Dendrio Rubus canescens, Rubus caucasicus, Rubus chamaemorus, poterium menendezii, Dendriopoterium pulidoi, Dryas Rubus corchorifolius, Rubus Crataegifolius, Rubus cuneifo drummondii, Dryas Octopetala, Duchesnea chrysantha, lius, Rubus deliciosus, Rubus divaricatus, Rubus ellipticus, Duchesnea indica, Erythrocoma triflora, Fallugia paradoxa, Rubus flagellaris, Rubus fruticosus, Rubus geoides, Rubus Filipendula multijuga Filipendula purpurea, Filipendula glabratus, Rubus glaucus, Rubus gunnianus, Rubus hawaien ulmaria, Filipendula vulgaris, Fragaria chiloensis, Fragaria sis, Rubus hawaiensisXRubus rosifolius, Rubus hispidus, daltoniana, Fragaria gracilis, Fragaria grandiflora, Rubus hochstetterorum, Rubus humulifolius, Rubus idaeus, Fragaria inumae, Fragaria moschata, Fragaria nilgerren Rubus lambertianus, Rubus lasiococcus, Rubus leucodermis, sis, Fragaria nipponica, Fragaria nubicola, Fragaria Orien Rubus lineatus, Rubus macraei, Rubus maximiformis, Rubus talis, Fragaria pentaphylla, Fragaria vesca, Fragaria vir minusculus, Rubus moorei, Rubus multibracteatus, Rubus giniana, Fragaria viridis, Fragariaxananassa, Fragaria sp. neomexicanus, Rubus nepalensis, Rubus messensis, Rubus CFRA 538, Fragaria sp., Geum andicola, Geum borisi, inivalis, Rubus niveus, Rubus nubigenus, Rubus Occidentalis, Geum bulgaricum, Geum calthifolium, Geum chiloense, Rubus odoratus, Rubus palmatus, Rubus parviflorus, Rubus Geum geniculatum, Geum heterocarpum, Geum macrophyl parvifolius, Rubus parvus, Rubus pectinellus, Rubus pedatus, lum, Geum montanum, Geum reptans, Geum rivale, Geum Rubus pedemontanus, Rubus pensilvanicus, Rubus phoenico schofieldii, Geum speciosum, Geum urbanum, Geum vernum, lasius, Rubus picticaulis, Rubus pubescens, Rubus rigidus, Geum sp. Chase 2507 K, Hagenia abyssinica, Horkelia Rubus robustus, Rubus roseus, Rubus rosifolius, Rubus sanc cuneata, Horkelia fisca, Ivesia gordoni, Kerria japonica, tus, Rubus sapidus, Rubus saxatilis, Rubus setosus, Rubus Leucosidea sericea, Marcetella maderensis, Marcetella spectabilis, Rubus sulcatus, Rubus tephrodes, Rubus trian moquiniana, Margyricarpus pinnatus, Margyricarpus Seto thus, Rubus tricolor, Rubus trifidus, Rubus trilobus, Rubus sus, Novosieversia glacialis, Oncostylus cockaynei, Oncostly trivialis, Rubus ulmifolius, Rubus ursinus, Rubus urticifolius, lus leiospermus, Polylepis australis, Polylepis besseri, Polyl Rubus vigorosus, Rubus sp. JPM-2004, Sanguisorba albi epis crista-galli, Polylepis hieronymi, Polylepis incana, flora, Sanguisorba alpina, Sanguisorba ancistroides, San Polylepis lanuginosa, Polylepis multijuga, Polylepis guisorba annua, Sanguisorba Canadensis, Sanguisorba fili neglecta, Polylepis pauta, Polylepis pepei, Polylepis quadri formis, Sanguisorba hakusanensis, Sanguisorba japonensis, juga, Polylepis racemosa, Polylepis reticulata, Polylepis Sanguisorba minor; Sanguisorba obtusa, Sanguisorba offici rugulosa, Polylepis sericea, Polylepis subsericans, Polylepis nalis, Sanguisorba parviflora, Sanguisorba stipulata, San tarapacana, Polylepis tomentella, Polylepis weberbaueri, guisorba tenuifolia, Sarcopoterium spinosum, Sibbaldia Potentilla anserina, Potentilla arguta, Potentilla bifurca, procumbens, Sieversia pentapetala, Sieversia pusilla, Potentilla chinensis, Potentilla dickinsii, Potentilla erecta, Taihangia rupestris, Tetraglochin cristatum, Waldsteinia Potentilla fragarioides, Potentilla fruticosa, Potentilla fragarioides, Waldsteinia geoides, Adenostoma fasciculatum, indica, Potentilla micrantha, Potentilla multifida, Potentilla Adenostoma sparsifolium, Aruncus dioicus, Cercocarpus nivea, Potentilla norvegica, Potentilla palustris, Potentilla betuloides, Cercocarpus ledifolius, Chamaebatiaria millefo peduncularis, Potentilla reptans, Potentilla salesoviana, lium, Chamaerhodos erecta, Gillenia stipulata, Gillenia tri Potentilla stenophylla, Potentilla tridentata, Rosa abietina, foliata, Holodiscus discolor, Holodiscus microphyllus, Rosa abyssinica, Rosa acicularis, Rosa agrestis, Rosa alba, Lyonothamnus floribundus, Neillia afinis, Neillia gracilis, Rosa albax Rosa corymbifera, Rosa altaica, Rosa arkansana, Neillia sinensis, Neillia sparsiflora, Neillia thibetica, Neillia Rosa arvensis, Rosa banksiae, Rosa beggeriana, Rosa thyrsiflora, Neillia uekii, Neviusia alabamensis, Physocarpus blanda, Rosa bracteata, Rosa brunonii, Rosa caesia, Rosa alternans, Physocarpus amurensis, Physocarpus capitatus, Californica, Rosa canina, Rosa Carolina, Rosa Chinensis, Physocarpus malvaceus, Physocarpus monogynus, Physo Rosa cinnamomea, Rosa columnifera, Rosa corymbifera, carpus opulifolius, Purshia tridentata, Rhodotypos scandens, Rosa cymosa, Rosa davurica, Rosa dumalis, Rosa ecae, Rosa Sorbaria arborea, Sorbaria sorbifolia, Spiraea betulifolia, eglanteria, Rosa elliptica, Rosa fedtschenkoana, Rosa foe Spiraea Cantoniensis, Spiraea densiflora, Spiraea japonica, US 2011/O 191902 A1 Aug. 4, 2011
Spiraea nipponica, Spiraeaxvanhouttei, Spiraea sp., Stepha the fruit of the method of the invention. It is not intended that nandra chinensis, Stephanandra incisa and Stephanandra “increased firmness” during, or after, post-harvest storage tanakae. means that a fruit becomes more firm than it was before 0128. A particularly preferred genus is Malus. post-harvest storage. 0129. Preferred Malus species include: Malus aldenhamii 0145 Similarly each of the other terms b) to g) above are Malus angustifolia, Malus asiatica, Malus baccata, Malus relative to control fruit under the same conditions. coronaria, Malus domestica, Malus doumeri, Malus floren 0146 The term “post-harvest storage' relates to storage of tina, Malus floribunda, Malus fisca, Malus halliana, Malus the fruit after harvesting from the plant/tree. honanensis, Malus hupehensis, Malus ioensis, Malus kan 0147 Typical post harvest storage conditions may include suensis, Malus mandshurica, Malus micromalus, Malus storage in controlled atmosphere, and/or modification oftem niedzwetzkyana, Malus Ombrophilia, Malus Orientalis, Malus perature (typically 0.5° C. to 3° C.) and/or application of prattii, Malus prunifolia, Malus pumila, Malus Sargentii, growth regulators (such as 1-MCP). Malus sieboldii, Malus sieversii, Malus Sylvestris, Malus tor 0148 Preferred post-harvest storage conditions depend on ingoides, Malus transitoria, Malus trilobata, Malus the region of growth and how long it is anticipated that the tSchonoskii, MalusXdomestica, MalusXdomesticaxMalus fruit need to be stored. sieversii, Malus Sylvestris, MalusXdomesticaXPyrus commu 0149 Exemplary post-harvest storage conditions for the nis, Malus xiaojinensis, Malus yunnanensis, Malus sp., purpose of the invention are 0.5°C. for 10 weeks Mespilus germanica, 0130. A particularly preferred plant species is Malus Polynucleotides and Fragments domestica. 0131 Methods of the invention that include producing 0150. The term “polynucleotide(s).' as used herein, means plants with reduced water loss are suitable for all fruit species. a single or double-stranded deoxyribonucleotide or ribo 0132 Methods of the invention that include producing nucleotide polymer of any length but preferably at least 15 plants with increased firmness are particularly suitable for nucleotides, and include as non-limiting examples, coding Malus species and fruit which don’t have a melting texture and non-coding sequences of a gene, sense and antisense when ripe such as Asian pear and non-melting peaches. sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified DETAILED DESCRIPTION naturally occurring DNA or RNA sequences, synthetic RNA 0133. In this specification where reference has been made and DNA sequences, nucleic acid probes, primers and frag to patent specifications, other external documents, or other mentS. Sources of information, this is generally for the purpose of 0151. A “fragment of a polynucleotide sequence pro providing a context for discussing the features of the inven vided herein is a Subsequence of contiguous nucleotides that tion. Unless specifically stated otherwise, reference to such is capable of specific hybridization to a target of interest, e.g., external documents is not to be construed as an admission that a sequence that is at least 15 nucleotides in length. The frag Such documents, or Such sources of information, in any juris ments of the invention comprise 15 nucleotides, preferably at diction, are prior art, or form part of the common general least 16 nucleotides, more preferably at least 17 nucleotides, knowledge in the art. more preferably at least 18 nucleotides, more preferably at 0134. The term “comprising as used in this specification least 19 nucleotides, more preferably at least 20 nucleotides, means "consisting at least in part of. When interpreting each more preferably at least 21 nucleotides, more preferably at statement in this specification that includes the term "com least 22 nucleotides, more preferably at least 23 nucleotides, prising, features other than that or those prefaced by the term more preferably at least 24 nucleotides, more preferably at may also be present. Related terms such as "comprise' and least 25 nucleotides, more preferably at least 26 nucleotides, “comprises are to be interpreted in the same manner. more preferably at least 27 nucleotides, more preferably at 0135 The invention provides methods and composition least 28 nucleotides, more preferably at least 29 nucleotides, for producing plants with fruit having increased post-harvest more preferably at least 30 nucleotides, more preferably at storage life. The fruit have at least one of the following char least 31 nucleotides, more preferably at least 32 nucleotides, acteristics: more preferably at least 33 nucleotides, more preferably at 0.136 a) increased firmness, least 34 nucleotides, more preferably at least 35 nucleotides, 0137 b) reduced water loss, more preferably at least 36 nucleotides, more preferably at 0138 c) reduced cell separation, least 37 nucleotides, more preferably at least 38 nucleotides, 0139 d) increased juiciness, more preferably at least 39 nucleotides, more preferably at 0140 e) increased crispiness, least 40 nucleotides, more preferably at least 41 nucleotides, 0141 f) increased waxiness, and more preferably at least 42 nucleotides, more preferably at 0142 g) reduced Susceptibility to necrophytic pathogens. least 43 nucleotides, more preferably at least 44 nucleotides, 0143. The terms, a) to g), are intended to be relative to more preferably at least 45 nucleotides, more preferably at terms. That is relative to the fruit of control plants under the least 46 nucleotides, more preferably at least 47 nucleotides, same conditions. more preferably at least 48 nucleotides, more preferably at 0144. For example a fruit with “increased firmness” is least 49 nucleotides, more preferably at least 50 nucleotides, more firm than a control fruit (or fruit of a control plant) more preferably at least 51 nucleotides, more preferably at Subjected to the same conditions during, or after, post-harvest least 52 nucleotides, more preferably at least 53 nucleotides, storage. Thus “increased firmness of the fruit of the method more preferably at least 54 nucleotides, more preferably at of the invention, is equivalent to “reduced softness” of the least 55 nucleotides, more preferably at least 56 nucleotides, fruit of the method of the invention, when the control plant more preferably at least 57 nucleotides, more preferably at softens more during or after post-harvest storage than does least 58 nucleotides, more preferably at least 59 nucleotides, US 2011/O 191902 A1 Aug. 4, 2011 more preferably at least 60 nucleotides, more preferably at the biological activity and/or provides three dimensional least 61 nucleotides, more preferably at least 62 nucleotides, structure of the polypeptide. The term may refer to a polypep more preferably at least 63 nucleotides, more preferably at tide, an aggregate of a polypeptide Such as a dimer or other least 64 nucleotides, more preferably at least 65 nucleotides, multimer, a fusion polypeptide, a polypeptide fragment, a more preferably at least 66 nucleotides, more preferably at polypeptide variant, or derivative thereof capable of perform least 67 nucleotides, more preferably at least 68 nucleotides, ing the above enzymatic activity. more preferably at least 69 nucleotides, more preferably at 0157. The term "isolated as applied to the polynucleotide least 70 nucleotides, more preferably at least 71 nucleotides, or polypeptide sequences disclosed herein is used to refer to more preferably at least 72 nucleotides, more preferably at sequences that are removed from their natural cellular envi least 73 nucleotides, more preferably at least 74 nucleotides, ronment. An isolated molecule may be obtained by any more preferably at least 75 nucleotides, more preferably at method or combination of methods including biochemical, least 76 nucleotides, more preferably at least 77 nucleotides, recombinant, and synthetic techniques. more preferably at least 78 nucleotides, more preferably at 0158. The term “recombinant” refers to a polynucleotide least 79 nucleotides, more preferably at least 80 nucleotides, sequence that is removed from sequences that Surround it in more preferably at least 81 nucleotides, more preferably at its natural context and/or is recombined with sequences that least 82 nucleotides, more preferably at least 83 nucleotides, are not present in its natural context. more preferably at least 84 nucleotides, more preferably at 0159. A “recombinant polypeptide sequence is produced least 85 nucleotides, more preferably at least 86 nucleotides, by translation from a “recombinant polynucleotide more preferably at least 87 nucleotides, more preferably at Sequence. least 88 nucleotides, more preferably at least 89 nucleotides, 0160 The term "derived from with respect to polynucle more preferably at least 90 nucleotides, more preferably at otides or polypeptides of the invention being derived from a least 91 nucleotides, more preferably at least 92 nucleotides, particular genera or species, means that the polynucleotide or more preferably at least 93 nucleotides, more preferably at polypeptide has the same sequence as a polynucleotide or least 94 nucleotides, more preferably at least 95 nucleotides, polypeptide found naturally in that genera or species. The more preferably at least 96 nucleotides, more preferably at polynucleotide or polypeptide, derived from a particular gen least 97 nucleotides, more preferably at least 98 nucleotides, era or species, may therefore be produced synthetically or more preferably at least 99 nucleotides, more preferably at recombinantly. least 100 nucleotides, more preferably at least 150 nucle otides, more preferably at least 200 nucleotides, more pref Variants erably at least 250 nucleotides, more preferably at least 300 nucleotides, more preferably at least 350 nucleotides, more 0.161. As used herein, the term “variant refers to poly preferably at least 400 nucleotides, more preferably at least nucleotide or polypeptide sequences different from the spe 450 nucleotides and most preferably at least 500 nucleotides cifically identified sequences, wherein one or more nucle of contiguous nucleotides of a polynucleotide disclosed. A otides oramino acid residues is deleted, Substituted, or added. fragment of a polynucleotide sequence can be used in anti Variants may be naturally occurring allelic variants, or non sense, RNA interference (RNAi), gene silencing, triple helix naturally occurring variants. Variants may be from the same or ribozyme technology, or as a primer, a probe, included in a or from other species and may encompass homologues, para microarray, or used in polynucleotide-based selection meth logues and orthologues. In certain embodiments, variants of ods of the invention. the inventive polypeptides and polypeptides possess biologi 0152 The term “primer' refers to a short polynucleotide, cal activities that are the same or similar to those of the usually having a free 3'OH group, that is hybridized to a inventive polypeptides or polypeptides. The term “variant” template and used for priming polymerization of a polynucle with reference to polypeptides and polypeptides encom otide complementary to the target. passes all forms of polypeptides and polypeptides as defined 0153. The term “probe' refers to a short polynucleotide herein. that is used to detect a polynucleotide sequence, that is complementary to the probe, in a hybridization-based assay. Polynucleotide Variants The probe may consist of a “fragment of a polynucleotide as defined herein. 0162 Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, more Polypeptides and Fragments preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more 0154 The term “polypeptide', as used herein, encom preferably at least 56%, more preferably at least 57%, more passes amino acid chains of any length but preferably at least preferably at least 58%, more preferably at least 59%, more 5 amino acids, including full-length proteins, in which amino preferably at least 60%, more preferably at least 61%, more acid residues are linked by covalent peptide bonds. Polypep preferably at least 62%, more preferably at least 63%, more tides of the present invention, or used in the methods of the preferably at least 64%, more preferably at least 65%, more invention, may be purified natural products, or may be pro preferably at least 66%, more preferably at least 67%, more duced partially or wholly using recombinant or synthetic preferably at least 68%, more preferably at least 69%, more techniques. preferably at least 70%, more preferably at least 71%, more 0155 The term may refer to a polypeptide, an aggregate of preferably at least 72%, more preferably at least 73%, more a polypeptide Such as a dimer or other multimer, a fusion preferably at least 74%, more preferably at least 75%, more polypeptide, a polypeptide fragment, a polypeptide variant, preferably at least 76%, more preferably at least 77%, more or derivative thereof. preferably at least 78%, more preferably at least 79%, more 0156. A “fragment of a polypeptide is a subsequence of preferably at least 80%, more preferably at least 81%, more the polypeptide that performs a function that is required for preferably at least 82%, more preferably at least 83%, more US 2011/O 191902 A1 Aug. 4, 2011
preferably at least 84%, more preferably at least 85%, more chance. Such sequence similarity with respect to polypep preferably at least 86%, more preferably at least 87%, more tides may be determined using the publicly available b12seq preferably at least 88%, more preferably at least 89%, more program from the BLAST suite of programs (version 2.2.5 preferably at least 90%, more preferably at least 91%, more November 2002) from NCBI (ftp://ftp.ncbi.nih.gov/blast/). preferably at least 92%, more preferably at least 93%, more 0170 The similarity of polynucleotide sequences may be preferably at least 94%, more preferably at least 95%, more examined using the following unix command line param preferably at least 96%, more preferably at least 97%, more eters: preferably at least 98%, and most preferably at least 99% 0171 b12seq -i nucleotideseq 1 - nucleotideseq2 -FF identity to a sequence of the present invention. Identity is -p thlastx found over a comparison window of at least 20 nucleotide 0172. The parameter -FF turns off filtering of low com positions, preferably at least 50 nucleotide positions, more plexity sections. The parameter -p selects the appropriate preferably at least 100 nucleotide positions, and most prefer algorithm for the pair of sequences. This program finds ably over the entire length of a polynucleotide of the inven regions of similarity between the sequences and for each Such tion. region reports an “E value' which is the expected number of 0163 Polynucleotide sequence identity can be determined times one could expect to see Such a match by chance in a in the following manner. The subject polynucleotide database of a fixed reference size containing random sequence is compared to a candidate polynucleotide sequence sequences. The size of this database is set by default in the using BLASTN (from the BLAST suite of programs, version b12seq program. For Small Evalues, much less than one, the 2.2.5 November 2002) in b12seq (Tatiana A. Tatusova, Tho E value is approximately the probability of such a random mas L. Madden (1999), "Blast 2 sequences—a new tool for match. comparing protein and nucleotide sequences. FEMS Micro 0173 Variant polynucleotide sequences preferably biol Lett. 174:247-250), which is publicly available from exhibit an E value of less than 1x10 more preferably less NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters than 1x10, more preferably less than 1x10', more pref ofb12seq are utilized except that filtering of low complexity erably less than 1x10', more preferably less than 1x10, parts should be turned off. more preferably less than 1x10', more preferably less than 0164. The identity of polynucleotide sequences may be 1x10, more preferably less than 1x10', more preferably examined using the following unix command line param less than 1x10, more preferably less than 1x10' more eters: preferably less than 1x10', more preferably less than 1x10 b12seq -i nucleotideseq 1 - nucleotideseq2 -F F-p blastin 80, more preferably less than 1x10' and most preferably less 0.165. The parameter -F F turns off filtering of low com than 1x10" when compared with any one of the specifi plexity sections. The parameter -p selects the appropriate cally identified sequences. algorithm for the pair of sequences. The b12seq program 0.174 Alternatively, variant polynucleotides of the present reports sequence identity as both the number and percentage invention, or used in the methods of the invention, hybridize of identical nucleotides in a line “Identities='. to the specified polynucleotide sequences, or complements 0166 Polynucleotide sequence identity may also be cal thereof under Stringent conditions. culated over the entire length of the overlap between a can 0.175. The term “hybridize under stringent conditions', didate and Subject polynucleotide sequences using global and grammatical equivalents thereof, refers to the ability of a sequence alignment programs (e.g. Needleman, S. B. and polynucleotide molecule to hybridize to a target polynucle Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A full otide molecule (such as a target polynucleotide molecule implementation of the Needleman-Wunsch global alignment immobilized on a DNA or RNA blot, such as a Southern blot algorithm is found in the needle program in the EMBOSS or Northern blot) under defined conditions oftemperature and package (Rice, P. Longden, I. and Bleasby, A. EMBOSS: The salt concentration. The ability to hybridize under stringent European Molecular Biology Open Software Suite, Trends in hybridization conditions can be determined by initially Genetics June 2000, vol 16, No 6. pp. 276-277) which can be hybridizing under less stringent conditions then increasing obtained from http://www.hgmp.mrc.ac.uk/Software/EM the stringency to the desired stringency. BOSS/. The European Bioinformatics Institute server also 0176 With respect to polynucleotide molecules greater provides the facility to perform EMBOSS-needle global than about 100 bases in length, typical stringent hybridization alignments between two sequences on line at http:/www.ebi. conditions are no more than 25 to 30°C. (for example, 10°C.) ac.uk/emboss/align/. below the melting temperature (Tm) of the native duplex (see 0167 Alternatively the GAP program may be used which generally, Sambrook et al., Eds, 1987, Molecular Cloning. A computes an optimal global alignment of two sequences Laboratory Manual, 2nd Ed. Cold Spring Harbor Press: without penalizing terminal gaps. GAP is described in the Ausubel et al., 1987, Current Protocols in Molecular Biology, following paper: Huang, X. (1994) On Global Sequence Greene Publishing.). Tm for polynucleotide molecules Alignment. Computer Applications in the BioSciences 10, greater than about 100 bases can be calculated by the formula 227-235. Tm=81.5+0.41% (G+C-log(Na+). (Sambrook et al., Eds, 0168 A preferred method for calculating polynucleotide 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. % sequence identity is based on aligning sequences to be Cold Spring Harbor Press; Bolton and McCarthy, 1962, compared using Clustal X (Jeanmougin et al., 1998, Trends PNAS 84:1390). Typical stringent conditions for polynucle Biochem. Sci. 23, 403-5.) otide of greater than 100 bases in length would be hybridiza 0169 Polynucleotide variants of the present invention also tion conditions such as prewashing in a solution of 6xSSC, encompass those which exhibit a similarity to one or more of 0.2% SDS: hybridizing at 65° C., 6xSSC, 0.2% SDS over the specifically identified sequences that is likely to preserve night; followed by two washes of 30 minutes each in lx SSC, the functional equivalence of those sequences and which 0.1% SDS at 65° C. and two washes of 30 minutes each in could not reasonably be expected to have occurred by random 0.2xSSC, 0.1% SDS at 65° C. US 2011/O 191902 A1 Aug. 4, 2011
0177. With respect to polynucleotide molecules having a least 67%, more preferably at least 68%, more preferably at length less than 100 bases, exemplary Stringent hybridization least 69%, more preferably at least 70%, more preferably at conditions are 5 to 10°C. below Tm. On average, the Tm of a least 71%, more preferably at least 72%, more preferably at polynucleotide molecule of length less than 100 bp is reduced least 73%, more preferably at least 74%, more preferably at by approximately (500/oligonucleotide length) C. least 75%, more preferably at least 76%, more preferably at 0.178 With respect to the DNA mimics known as peptide least 77%, more preferably at least 78%, more preferably at nucleic acids (PNAS) (Nielsen et al., Science. 1991 Dec. 6; least 79%, more preferably at least 80%, more preferably at 254(5037): 1497-500) Tm values are higher than those for least 81%, more preferably at least 82%, more preferably at DNA-DNA or DNA-RNA hybrids, and can be calculated least 83%, more preferably at least 84%, more preferably at using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov. 1; 26(21):5004-6. Exemplary stringent least 85%, more preferably at least 86%, more preferably at hybridization conditions for a DNA-PNA hybrid having a least 87%, more preferably at least 88%, more preferably at length less than 100 bases are 5 to 10° C. below the Tm. least 89%, more preferably at least 90%, more preferably at 0179 Variant polynucleotides of the present invention, or least 91%, more preferably at least 92%, more preferably at used in the methods of the invention, also encompasses poly least 93%, more preferably at least 94%, more preferably at nucleotides that differ from the sequences of the invention but least 95%, more preferably at least 96%, more preferably at that, as a consequence of the degeneracy of the genetic code, least 97%, more preferably at least 98%, and most preferably encode a polypeptide having similar activity to a polypeptide at least 99% identity to a sequences of the present invention. encoded by a polynucleotide of the present invention. A Identity is found over a comparison window of at least 20 sequence alteration that does not change the amino acid amino acid positions, preferably at least 50 amino acid posi sequence of the polypeptide is a “silent variation”. Except for tions, more preferably at least 100 amino acid positions, and ATG (methionine) and TGG (tryptophan), other codons for most preferably over the entire length of a polypeptide of the the same amino acid may be changed by art recognized tech invention. Polypeptide sequence identity can be determined niques, e.g., to optimize codon expression in a particular host in the following manner. The Subject polypeptide sequence is organism. compared to a candidate polypeptide sequence using 0180 Polynucleotide sequence alterations resulting in BLASTP (from the BLAST suite of programs, version 2.2.5 conservative Substitutions of one or several amino acids in the November 2002) in b12seq, which is publicly available encoded polypeptide sequence without significantly altering from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default param its biological activity are also included in the invention. A eters of b12seq are utilized except that filtering of low com skilled artisan will be aware of methods for making pheno plexity regions should be turned off. typically silent amino acid Substitutions (see, e.g., Bowie et 0.184 Polypeptide sequence identity may also be calcu al., 1990, Science 247, 1306). lated over the entire length of the overlap between a candidate 0181 Variant polynucleotides due to silent variations and and Subject polynucleotide sequences using global sequence conservative Substitutions in the encoded polypeptide alignment programs. EMBOSS-needle (available at http:/ sequence may be determined using the publicly available www.ebi.ac.uk/emboss/align/) and GAP (Huang, X. (1994) b12seq program from the BLAST suite of programs (version On Global Sequence Alignment. Computer Applications in 2.2.5 November 2002) from NCBI (ftp://ftp.ncbi.nih.gov/ the Biosciences 10, 227-235.) as discussed above are also blast?) via the tblastX algorithm as previously described. Suitable global sequence alignment programs for calculating 0182. The function of a variant polynucleotide or polypep polypeptide sequence identity. tide of the invention, or used in the methods of the invention, 0185. A preferred method for calculating polypeptide % as a polygalacturonase may be assessed for example by sequence identity is based on aligning sequences to be com expressing Such a sequence in yeast and testing activity of the pared using Clustal X (Jeanmougin et al., 1998, Trends Bio encoded protein as previously described for cell wall related chem. Sci. 23, 403-5.) proteins (van Rensberg etal 1994; Saladie etal 2006). Func 0186 Polypeptide variants of the present invention, or tion of a variant may also be tested for its ability to alter used in the methods of the invention, also encompass those polygalacturonase activity in plants, as described in (Hellens which exhibit a similarity to one or more of the specifically et al 2005). The function of variants in altering post-harvest identified sequences that is likely to preserve the functional storage life may be tested by methods described in this speci equivalence of those sequences and which could not reason fication (e.g., in the Examples section) and by other methods ably be expected to have occurred by random chance. Such known to those skilled in the art. sequence similarity with respect to polypeptides may be determined using the publicly available b12seq program from Polypeptide Variants the BLAST suite of programs (version 2.2.5 November 0183 The term “variant” with reference to polypeptides 2002) from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The simi encompasses naturally occurring, recombinantly and Syn larity of polypeptide sequences may be examined using the thetically produced polypeptides. Variant polypeptide following unix command line parameters: sequences preferably exhibit at least 50%, more preferably at 0187. b12seq-i peptideseq 1 -peptideseq2-FF-p blastp least 51%, more preferably at least 52%, more preferably at 0188 Variant polypeptide sequences preferably exhibitan least 53%, more preferably at least 54%, more preferably at Evalue of less than 1x10 more preferably less than 1x10, least 55%, more preferably at least 56%, more preferably at more preferably less than 1x10', more preferably less than least 57%, more preferably at least 58%, more preferably at 1x10', more preferably less than 1x10, more preferably least 59%, more preferably at least 60%, more preferably at less than 1x10', more preferably less than 1x10", more least 61%, more preferably at least 62%, more preferably at preferably less than 1x10", more preferably less than 1x10 least 63%, more preferably at least 64%, more preferably at so, more preferably less than 1x10', more preferably less least 65%, more preferably at least 66%, more preferably at than 1x10', more preferably less than 1x10', more pref US 2011/O 191902 A1 Aug. 4, 2011 erably less than 1x10' and most preferably 1x10' when 0199 The term “noncoding region” refers to untranslated compared with any one of the specifically identified sequences that are upstream of the translational start site and Sequences. downstream of the translational stop site. These sequences 0189 The parameter -F F turns off filtering of low com are also referred to respectively as the 5' UTR and the 3' UTR. plexity sections. The parameter -p selects the appropriate These regions include elements required for transcription algorithm for the pair of sequences. This program finds initiation and termination and for regulation of translation regions of similarity between the sequences and for each Such efficiency. region reports an “E value' which is the expected number of 0200 Terminators are sequences, which terminate tran times one could expect to see Such a match by chance in a Scription, and are found in the 3' untranslated ends of genes database of a fixed reference size containing random downstream of the translated sequence. Terminators are sequences. For Small E Values, much less than one, this is important determinants of mRNA stability and in some cases approximately the probability of Such a random match. have been found to have spatial regulatory functions. 0190. Conservative substitutions of one or several amino 0201 The term “promoter refers to nontranscribed cis acids of a described polypeptide sequence without signifi regulatory elements upstream of the coding region that regu cantly altering its biological activity are also included in the late gene transcription. Promoters comprise cis-initiator ele invention. A skilled artisan will be aware of methods for ments which specify the transcription initiation site and making phenotypically silent amino acid substitutions (see, conserved boxes such as the TATA box, and motifs that are e.g., Bowie et al., 1990, Science 247, 1306). bound by transcription factors. 0202 A promoter may be homologous with respect to the Constructs, Vectors and Components Thereof. polynucleotide to be expressed. This means that the promoter and polynucleotide are found operably linked in nature. 0191 The term “genetic construct” refers to a polynucle 0203 Alternatively the promoter may be heterologous otide molecule, usually double-stranded DNA, which may with respect to the polynucleotide to be expressed. This have inserted into it another polynucleotide molecule (the means that the promoter and the polynucleotide are not found insert polynucleotide molecule) Such as, but not limited to, a operably linked in nature. cDNA molecule. A genetic construct may contain the neces 0204. A “transgene' is a polynucleotide that is taken from sary elements that permit transcribing the insert polynucle one organism and introduced into a different organism by otide molecule, and, optionally, translating the transcript into transformation. The transgene may be derived from the same a polypeptide. The insert polynucleotide molecule may be species or from a different species as the species of the organ derived from the host cell, or may be derived from a different cell or organism and/or may be a recombinant polynucle ism into which the transgene is introduced. otide. Once inside the host cell the genetic construct may 0205 An “inverted repeat' is a sequence that is repeated, become integrated in the host chromosomal DNA. The where the second half of the repeat is in the complementary genetic construct may be linked to a vector. Strand, e.g., 0.192 The term “vector” refers to a polynucleotide mol ecule, usually double stranded DNA, which is used to trans (5') GATCTA . . TAGATC (3') port the genetic construct into a host cell. The vector may be (3') CTAGAT . . ATCTAG (5') capable of replication in at least one additional host system, Such as E. coli. 0206 Read-through transcription will produce a transcript 0193 The term “expression construct” refers to a genetic that undergoes complementary base-pairing to form a hairpin construct that includes the necessary elements that permit structure provided that there is a 3-5 bp spacer between the transcribing the insert polynucleotide molecule, and, option repeated regions. ally, translating the transcript into a polypeptide. An expres sion construct typically comprises in a 5' to 3' direction: Host Cells 0194 a) a promoter functional in the host cell into 0207 Host cells may be derived from, for example, bac which the construct will be transformed, terial, fungal, insect, mammalian or plant organisms. 0.195 b) the polynucleotide to be expressed, and 0208. A “transgenic plant” refers to a plant which contains 0.196 c) a terminator functional in the host cell into new genetic material as a result of genetic manipulation or which the construct will be transformed. transformation. The new genetic material may be derived 0197) The term “coding region' or “open reading frame' from a plant of the same species as the resulting transgenic (ORF) refers to the sense strand of a genomic DNA sequence plant or from a different species. or a cDNA sequence that is capable of producing a transcrip 0209. The applicants have surprisingly shown that plants tion product and/or a polypeptide under the control of appro transformed to reduce expression of the polypeptide of SEQ priate regulatory sequences. The coding sequence is identi ID NO: 1, produce fruit with increased post-harvest storage fied by the presence of a 5' translation start codon and a 3' life. translation stop codon. When inserted into a genetic con 0210. The plants have, or are capable of producing, fruit struct, a “coding sequence' is capable of being expressed with the following characteristics: when it is operably linked to promoter and terminator 0211 a) increased firmness, Sequences. 0212 b) reduced water loss, 0198 “Operably-linked' means that the sequenced to be 0213 c) reduced cell separation, expressed is placed under the control of regulatory elements 0214 d) increased juiciness, that include promoters, tissue-specific regulatory elements, 0215 e) increased crispiness, temporal regulatory elements, enhancers, repressors and ter 0216 f) increased waxiness, and minators. 0217 g) reduced susceptibility to necrophytic pathogens. US 2011/O 191902 A1 Aug. 4, 2011
0218. The invention provides expression constructs suit result, it may be necessary to utilise a sequence identical (or able for reducing the expression of the polypeptide of SEQID at least highly similar) to that in the plant, for which reduced NO: 1 or variants thereof. The invention also provides plant expression is desired. For these reasons among others, it is cells and plants comprising the expression constructs. desirable to be able to identify and isolate orthologues of a 0219. The invention also provides methods for producing, particular gene in several different plant species. and selecting plants with increased post-harvest storage life, 0226 Variants (including orthologues) may be identified relative to suitable control plants. by the methods described. 0220 Suitable control plants include non-transformed plants of the same species or variety or plants transformed Methods for Identifying Variants with control constructs. Physical Methods Methods for Isolating or Producing Polynucleotides 0227 Variant polypeptides may be identified using PCR 0221) The polynucleotide molecules of the invention can based methods (Mullis et al., Eds. 1994 The Polymerase be isolated by using a variety of techniques known to those of Chain Reaction, Birkhauser). Typically, the polynucleotide ordinary skill in the art. By way of example, Such polypep sequence of a primer, useful to amplify variants of polynucle tides can be isolated through use of the polymerase chain otide molecules of the invention by PCR, may be based on a reaction (PCR) described in Mullis et al., Eds. 1994 The sequence encoding a conserved region of the corresponding Polymerase Chain Reaction, Birkhauser, incorporated herein amino acid sequence. by reference. The polypeptides of the invention can be ampli 0228. Alternatively library screening methods, well fied using primers, as defined herein, derived from the poly known to those skilled in the art, may be employed (Sam nucleotide sequences of the invention. brook et al., Molecular Cloning: A Laboratory Manual, 2nd 0222 Further methods for isolating polynucleotides of the Ed. Cold Spring Harbor Press, 1987). When identifying vari invention include use of all, or portions of the polypeptides ants of the probe sequence, hybridization and/or wash Strin having the sequence set forth herein as hybridization probes. gency will typically be reduced relatively to when exact The technique of hybridizing labelled polynucleotide probes sequence matches are sought. to polynucleotides immobilized on Solid Supports such as 0229 Polypeptide variants may also be identified by nitrocellulose filters or nylon membranes, can be used to physical methods, for example by Screening expression screen the genomic or cDNA libraries. Exemplary hybridiza libraries using antibodies raised against polypeptides of the tion and wash conditions are: hybridization for 20 hours at invention (Sambrook et al., Molecular Cloning: A Laboratory 65° C. in 5.0xSSC, 0.5% sodium dodecyl sulfate, 1xDen Manual, 2nd Ed. Cold Spring Harbor Press, 1987) or by hardt's solution; washing (three washes of twenty minutes identifying polypeptides from natural sources with the aid of each at 55°C.) in 1.0xSSC, 1% (w/v) sodium dodecyl sulfate, Such antibodies. and optionally one wash (for twenty minutes) in 0.5xSSC, 1% (w/v) sodium dodecyl sulfate, at 60° C. An optional further Computer Based Methods wash (for twenty minutes) can be conducted under conditions 0230. The variant sequences of the invention, including of 0.1xSSC, 1% (w/v) sodium dodecyl sulfate, at 60° C. both polynucleotide and polypeptide variants, may also be 0223) The polynucleotide fragments of the invention may identified by computer-based methods well-known to those be produced by techniques well-known in the art Such as skilled in the art, using public domain sequence alignment restriction endonuclease digestion, oligonucleotide synthesis algorithms and sequence similarity search tools to search and PCR amplification. sequence databases (public domain databases include Gen 0224. A partial polynucleotide sequence may be used, in bank, EMBL, Swiss-Prot, PIR and others). See, e.g., Nucleic methods well-known in the art to identify the corresponding Acids Res. 29: 1-10 and 11-16, 2001 for examples of online full length polynucleotide sequence. Such methods include resources. Similarity searches retrieve and align target PCR-based methods, 5'RACE (Frohman MA, 1993, Methods sequences for comparison with a sequence to be analyzed Enzymol. 218: 340-56) and hybridization-based method, (i.e., a query sequence). Sequence comparison algorithms use computer/database-based methods. Further, by way of scoring matrices to assign an overall score to each of the example, inverse PCR permits acquisition of unknown alignments. sequences, flanking the polynucleotide sequences disclosed 0231. An exemplary family of programs useful for identi herein, starting with primers based on a known region (Triglia fying variants in sequence databases is the BLAST Suite of et al., 1998, Nucleic Acids Res 16, 8186, incorporated herein programs (version 2.2.5 November 2002) including by reference). The method uses several restriction enzymes to BLASTN, BLASTP, BLASTX, tRLASTN and tRLASTX, generate a suitable fragment in the known region of a gene. which are publicly available from (ftp://ftp.ncbi.nih.gov/ The fragment is then circularized by intramolecular ligation blast/) or from the National Center for Biotechnology Infor and used as a PCR template. Divergent primers are designed mation (NCBI), National Library of Medicine, Building 38A, from the known region. In order to physically assemble full Room 8N805, Bethesda, Md. 20894 USA. The NCBI server length clones, standard molecular biology approaches can be also provides the facility to use the programs to Screen a utilized (Sambrook et al., Molecular Cloning: A Laboratory number of publicly available sequence databases. BLASTN Manual, 2nd Ed. Cold Spring Harbor Press, 1987). compares a nucleotide query sequence against a nucleotide 0225. It may be beneficial, when producing a transgenic sequence database. BLASTP compares an amino acid query plant from a particular species, to transform Such a plant with sequence against a protein sequence database. BLASTX a sequence or sequences derived from that species. The ben compares a nucleotide query sequence translated in all read efit may be to alleviate public concerns regarding cross-spe ing frames against a protein sequence database. tBLASTN cies transformation in generating transgenic organisms. compares a protein query sequence against a nucleotide Additionally when down-regulation of a gene is the desired sequence database dynamically translated in all reading US 2011/O 191902 A1 Aug. 4, 2011
frames. tBLASTX compares the six-frame translations of a proteins or to determine which known domain(s) are present nucleotide query sequence against the six-frame translations in the sequence (Falquet et al., 2002, Nucleic Acids Res. 30. of a nucleotide sequence database. The BLAST programs 235). Prosearch is a tool that can search SWISS-PROT and may be used with default parameters or the parameters may EMBL databases with a given sequence pattern or signature. be altered as required to refine the screen. 0238. The function of a variant polynucleotide of the 0232. The use of the BLAST family of algorithms, includ invention as encoding phloretin glycosyltransferases can be ing BLASTN, BLASTP, and BLASTX, is described in the tested for the activity, or can be tested for their capability to publication of Altschul et al., Nucleic Acids Res. 25: 3389 alter phlorizin content in plants by methods described in the 3402, 1997. examples section herein. 0233. The “hits” to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, Methods for Isolating Polypeptides BLASTX, thBLASTN, tBLASTX, or a similar algorithm, 0239. The polypeptides of the invention, or used in the align and identify similar portions of sequences. The hits are methods of the invention, including variant polypeptides, arranged in order of the degree of similarity and the length of may be prepared using peptide synthesis methods well known sequence overlap. Hits to a database sequence generally rep in the art such as direct peptide synthesis using solid phase resent an overlap over only a fraction of the sequence length techniques (e.g. Stewart et al., 1969, in Solid-Phase Peptide of the queried sequence. Synthesis, WH Freeman Co., San Francisco Calif., or auto 0234. The BLASTN, BLASTP, BLASTX, thBLASTN and mated synthesis, for example using an Applied Biosystems tBLASTX algorithms also produce “Expect values for 431A Peptide Synthesizer (Foster City, Calif.). Mutated alignments. The Expect value (E) indicates the number of hits forms of the polypeptides may also be produced during Such one can "expect to see by chance when searching a database syntheses. of the same size containing random contiguous sequences. 0240. The polypeptides and variant polypeptides of the The Expect value is used as a significance threshold for deter invention, or used in the methods of the invention, may also be mining whether the hit to a database indicates true similarity. purified from natural Sources using a variety of techniques For example, an E value of 0.1 assigned to a polynucleotide that are well known in the art (e.g. Deutscher, 1990, Ed, hit is interpreted as meaning that in a database of the size of Methods in Enzymology, Vol. 182, Guide to Protein Purifi the database screened, one might expect to see 0.1 matches cation.). over the aligned portion of the sequence with a similar score 0241 Alternatively the polypeptides and variant polypep simply by chance. For sequences having an Evalue of 0.01 or tides of the invention, or used in the methods of the invention, less over aligned and matched portions, the probability of may be expressed recombinantly in Suitable host cells and finding a match by chance in that database is 1% or less using separated from the cells as discussed below. the BLASTN, BLASTP. BLASTX, tRLASTN or tRLASTX algorithm. Methods for Producing Constructs and Vectors 0235 Multiple sequence alignments of a group of related sequences can be carried out with CLUSTALW (Thompson, 0242. The genetic constructs of the present invention com J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTALW. prise one or more polynucleotide sequences of the invention improving the sensitivity of progressive multiple sequence and/or polynucleotides encoding polypeptides of the inven alignment through sequence weighting, positions-specific tion, and may be useful for transforming, for example, bac gap penalties and weight matrix choice. Nucleic Acids terial, fungal, insect, mammalian or plant organisms. The Research, 22:4673-4680, http://www-igbmc.ustrasbg.fr/ genetic constructs of the invention are intended to include BioInfo/ClustalW/Top.html) or TCOFFEE (Cedric Notre expression constructs as herein defined. dame, Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel 0243 Methods for producing and using genetic constructs method for fast and accurate multiple sequence alignment, J. and vectors are well known in the art and are described Mol. Biol. (2000) 302: 205-217)) or PILEUP, which uses generally in Sambrook et al., Molecular Cloning: A Labora progressive, pairwise alignments. (Feng and Doolittle, 1987. tory Manual, 2nd Ed. Cold Spring Harbor Press, 1987: J. Mol. Evol. 25.351). Ausubel et al., Current Protocols in Molecular Biology, 0236 Pattern recognition software applications are avail Greene Publishing, 1987). able for finding motifs or signature sequences. For example, MEME (Multiple Em for Motif Elicitation) finds motifs and Methods for Producing Host Cells Comprising Polynucle signature sequences in a set of sequences, and MAST (Motif otides, Constructs or Vectors Alignment and Search Tool) uses these motifs to identify 0244. The invention provides a host cell which comprises similar or the same motifs in query sequences. The MAST a genetic construct or vector of the invention. results are provided as a series of alignments with appropriate 0245 Host cells comprising genetic constructs, such as statistical data and a visual overview of the motifs found. expression constructs, of the invention are useful in methods MEME and MAST were developed at the University of Cali well known in the art (e.g. Sambrook et al., Molecular Clon fornia, San Diego. ing: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 0237 PROSITE (Bairoch and Bucher, 1994, Nucleic 1987; Ausubel et al., Current Protocols in Molecular Biology, Acids Res. 22, 3583; Hofmann et al., 1999, Nucleic Acids Greene Publishing, 1987) for recombinant production of Res. 27, 215) is a method of identifying the functions of polypeptides of the invention. Such methods may involve the uncharacterized proteins translated from genomic or cDNA culture of host cells in an appropriate medium in conditions sequences. The PROSITE database (www.expasy.org/pros suitable for or conducive to expression of a polypeptide of the ite) contains biologically significant patterns and profiles and invention. The expressed recombinant polypeptide, which is designed so that it can be used with appropriate computa may optionally be secreted into the culture, may then be tional tools to assign a new sequence to a known family of separated from the medium, host cells or culture medium by US 2011/O 191902 A1 Aug. 4, 2011
methods well known in the art (e.g. Deutscher, Ed, 1990, are active in most plant tissues, and recombinant promoters. Methods in Enzymology, Vol 182, Guide to Protein Purifica Choice of promoter will depend upon the temporal and spatial tion). expression of the cloned polynucleotide, so desired. The pro moters may be those normally associated with a transgene of Methods for Producing Plant Cells and Plants Comprising interest, or promoters which are derived from genes of other Constructs and Vectors plants, viruses, and plant pathogenic bacteria and fungi. Those skilled in the art will, without undue experimentation, 0246 The invention further provides plant cells which be able to select promoters that are suitable for use in modi comprise a genetic construct of the invention, and plant cells fying and modulating plant traits using genetic constructs modified to alter expression of a polynucleotide or polypep comprising the polynucleotide sequences of the invention. tide of the invention, or used in the methods of the invention. Examples of constitutive plant promoters include the CaMV Plants comprising Such cells also forman aspect of the inven 35S promoter, the nopaline synthase promoter and the tion. octopine synthase promoter, and the Ubi 1 promoter from 0247 Alteration of post-harvest storage characteristics maize. Plant promoters which are active in specific tissues, may be altered in a plant through methods of the invention. respond to internal developmental signals or external abiotic Such methods may involve the transformation of plant cells or biotic stresses are described in the scientific literature. and plants, with a construct designed to alter expression of a Exemplary promoters are described, e.g., in WO 02/00894, polynucleotide or polypeptide which modulates post-harvest which is herein incorporated by reference. storage characteristics in Such plants. Such methods also 0253 Exemplary terminators that are commonly used in include the transformation of plant cells and plants with a plant transformation genetic construct include, e.g., the cau combination of the construct of the invention and one or more liflower mosaic virus (CaMV) 35S terminator, the Agrobac other constructs designed to alter expression of one or more terium tumefacizens nopaline synthase or octopine synthase polynucleotides or polypeptides which modulate post-har terminators, the Zea mays Zein gene terminator, the Oryza Vest storage characteristics in plants. sativa ADP-glucose pyrophosphorylase terminator and the 0248 Methods for transforming plant cells, plants and Solanum tuberosum PI-II terminator. portions thereof with polypeptides are described in Draper et 0254. Selectable markers commonly used in plant trans al., 1988, Plant Genetic Transformation and Gene Expres formation include the neomycin phophotransferase II gene sion. A Laboratory Manual. Blackwell Sci. Pub. Oxford, p. (NPT II) which confers kanamycin resistance, the aadA gene, 365; Potrykus and Spangenburg, 1995, Gene Transfer to which confers spectinomycin and streptomycin resistance, Plants. Springer-Verlag, Berlin.; and Gelvin et al., 1993, Plant the phosphinothricin acetyl transferase (bar gene) for Ignite Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A (AgrEvo) and Basta (Hoechst) resistance, and the hygromy review of transgenic plants, including transformation tech cin phosphotransferase gene (hpt) for hygromycin resistance. niques, is provided in Galun and Breiman, 1997, Transgenic 0255 Use of genetic constructs comprising reporter genes Plants. Imperial College Press, London. (coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and/or a visible signal Methods for Genetic Manipulation of Plants (e.g., luciferase, GUS, GFP) which may be used for promoter 0249. A number of plant transformation strategies are expression analysis in plants and plant tissues are also con available (e.g. Birch, 1997, Ann Rev Plant Phys Plant Mol templated. The reporter gene literature is reviewed in Her Biol, 48, 297, Hellens R P. et al (2000) Plant Mol Biol 42: rera-Estrella et al., 1993, Nature 303, 209, and Schrott, 1995, 819-32, Hellens R et al Plant Meth 1: 13). For example, In: Gene Transfer to Plants (Potrykus, T., Spangenberg. Eds) strategies may be designed to increase expression of a poly Springer Verlag. Berline, pp. 325-336. nucleotide/polypeptide in a plant cell, organ and/or at a par 0256 Gene silencing strategies may be focused on the ticular developmental stage where/when it is normally gene itself or regulatory elements which effect expression of expressed or to ectopically express a polynucleotide? the encoded polypeptide. “Regulatory elements' is used here polypeptide in a cell, tissue, organ and/or at a particular in the widest possible sense and includes other genes which developmental stage which/when it is not normally interact with the gene of interest. expressed. The expressed polynucleotide?polypeptide may be 0257 Genetic constructs designed to decrease or silence derived from the plant species to be transformed or may be the expression of a polynucleotide/polypeptide of the inven derived from a different plant species. tion may include an antisense copy of a polynucleotide of the 0250 Transformation strategies may be designed to invention. In such constructs the polynucleotide is placed in reduce expression of a polynucleotide/polypeptide in a plant an antisense orientation with respect to the promoter and cell, tissue, organ or at a particular developmental stage terminator. which/when it is normally expressed. Such strategies are 0258 An “antisense” polynucleotide is obtained by known as gene silencing strategies. inverting a polynucleotide or a segment of the polynucleotide 0251 Genetic constructs for expression of genes in trans so that the transcript produced will be complementary to the genic plants typically include promoters for driving the mRNA transcript of the gene, e.g., expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detect presence of the genetic construct in the transformed plant. 5' GATCTA 3 '' (coding 3 CTAGAT 5" (antisense Strand) 0252. The promoters suitable for use in the constructs of Strand) this invention are functional in a cell, tissue or organ of a 3 CUAGAU S" mRNA 5' GAUCUCG 3' antisense RNA monocot or dicot plant and include cell-, tissue- and organ specific promoters, cell cycle specific promoters, temporal 0259 Genetic constructs designed for gene silencing may promoters, inducible promoters, constitutive promoters that also include an inverted repeat. An inverted repeat is a US 2011/O 191902 A1 Aug. 4, 2011
sequence that is repeated where the second half of the repeat 522); brassica (U.S. Pat. Nos. 5,188,958; 5.463,174 and is in the complementary Strand, e.g., 5,750.871); cereals (U.S. Pat. No. 6,074,877); pear (Matsuda et al., 2005, Plant Cell Rep. 24(1):45-51); Prunus (Rameshet al., 2006 Plant Cell Rep. 25(8):821-8; Song and Sink 2005 5 - GATCTA . . TAGATC-3' Plant Cell Rep. 2006: 25(2):117-23; Gonzalez Padilla et al., 3 - CTAGAT . . ATCTAG-5 2003 Plant Cell Rep. 22(1):38-45); strawberry (Oosumiet al., 0260 The transcript formed may undergo complementary 2006 Planta. 223(6):1219-30; Folta et al., 2006 Planta April base pairing to form a hairpin structure. Usually a spacer of at 14; PMID: 16614818), rose (Liet al., 2003), Rubus (Graham least 3-5bp between the repeated region is required to allow et al., 1995 Methods Mol Biol. 1995; 44:129-33), tomato hairpin formation. Constructs including Such invented repeat (Dan et al., 2006, Plant Cell Reports V25:432-441), apple sequences may be used in RNA interference (RNAi) and (Yao et al. 1995, Plant Cell Rep. 14, 407-412) and Actinidia therefore can be referred to as RNAi constructs. eriantha (Wang et al., 2006, Plant Cell Rep. 25.5: 425-31). 0261) Another silencing approach involves the use of a Transformation of other species is also contemplated by the Small antisense RNA targeted to the transcript equivalent to invention. Suitable methods and protocols are available in the an miRNA (Llave et al., 2002, Science 297, 2053). Use of scientific literature. Such small antisense RNA corresponding to polynucleotide 0268. Several further methods known in the art may be of the invention is expressly contemplated. employed to alter expression of activity of a nucleotide and/or 0262 The term genetic construct as used herein also polypeptide of the invention. Such methods include but are includes Small antisense RNAS and other Such polypeptides not limited to Tilling (Tillet al., 2003, Methods Mol Biol, 2%, effecting gene silencing. 205), so called “Deletagene' technology (Li et al., 2001, 0263 Transformation with an expression construct, as Plant Journal 27(3), 235) and the use of artificial transcription herein defined, may also result in gene silencing through a factors such as synthetic Zinc finger transcription factors. (e.g. process known as sense Suppression (e.g. Napoli et al., 1990, Jouvenot et al., 2003, Gene Therapy 10, 513). Additionally Plant Cell 2, 279; de Carvalho Niebel et al., 1995, Plant Cell, antibodies or fragments thereof, targeted to a particular 7, 347). In some cases sense Suppression may involve over polypeptide may also be expressed in plants to modulate the expression of the whole or a partial coding sequence but may activity of that polypeptide (Jobling et al., 2003, Nat. Bio also involve expression of non-coding region of the gene, technol., 21 (1),35). Transposon tagging approaches may also such as an intron or a 5" or 3' untranslated region (UTR). be applied. Additionally peptides interacting with a polypep Chimeric partial sense constructs can be used to coordinately tide of the invention may be identified through technologies silence multiple genes (Abbott et al., 2002, Plant Physiol. Such as phase-display (Dyax Corporation). Such interacting 128(3): 844-53; Jones et al., 1998, Planta 204: 499-505). The peptides may be expressed in or applied to a plant to affect use of such sense Suppression strategies to silence the expres activity of a polypeptide of the invention. Use of each of the sion of a polynucleotide of the invention is also contemplated. above approaches in alteration of expression of a nucleotide 0264. The polynucleotide inserts in genetic constructs and/or polypeptide of the invention is specifically contem designed for gene silencing may correspond to coding plated. sequence and/or non-coding sequence, Such as promoter and/ 0269. The terms “to alter expression of and “altered or intron and/or 5' or 3' UTR sequence, of the corresponding expression of a polynucleotide or polypeptide of the inven gene. tion, or used in the methods of the invention, are intended to 0265. Other gene silencing strategies include dominant encompass the situation where genomic DNA corresponding negative approaches and the use of ribozyme constructs to a polynucleotide of the invention is modified thus leading (McIntyre, 1996, Transgenic Res, 5, 257). to altered expression of a polynucleotide or polypeptide of the 0266 Pre-transcriptional silencing may be brought about invention. Modification of the genomic DNA may be through through mutation of the gene itself or its regulatory elements. genetic transformation or other methods known in the art for Such mutations may include point mutations, frameshifts, inducing mutations. The “altered expression” can be related insertions, deletions and Substitutions. to an increase or decrease in the amount of messenger RNA 0267. The following are representative publications dis and/or polypeptide produced and may also result in altered closing genetic transformation protocols that can be used to activity of a polypeptide due to alterations in the sequence of genetically transform the following plant species: Rice (Alam a polynucleotide and polypeptide produced. et al., 1999, Plant Cell Rep. 18, 572); apple (Yao et al., 1995, Methods of Selecting Plants Plant Cell Reports 14,407-412); maize (U.S. Pat. Nos. 5,177, 010 and 5,981,840); wheat (Ortiz et al., 1996, Plant Cell Rep. 0270 Methods are also provided for selecting plants with 15, 1996, 877); tomato (U.S. Pat. No. 5,159,135); potato altered post-harvest storage characteristics. Such methods (Kumar et al., 1996 Plant J. 9, : 821); cassaya (Liet al., 1996 involve testing of plants for altered for the expression of a Nat. Biotechnology 14, 736): lettuce (Michelmore et al., polynucleotide or polypeptide of the invention, or disclosed 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, herein. Such methods may be applied at a young age or early Science 227, 1229): cotton (U.S. Pat. Nos. 5,846,797 and developmental stage when the altered post-harvest storage 5,004,863); grasses (U.S. Pat. Nos. 5,187,073 and 6,020, characteristics may not necessarily be easily measurable. 539); peppermint (Niu et al., 1998, Plant Cell Rep. 17, 165); 0271 The expression of a polynucleotide, such as a mes citrus plants (Pena et al., 1995, Plant Sci. 104, 183); caraway senger RNA, is often used as an indicator of expression of a (Krens et al., 1997, Plant Cell Rep. 17, 39); banana (U.S. Pat. corresponding polypeptide. Exemplary methods for measur No. 5,792,935); soybean (U.S. Pat. Nos. 5,416,011; 5,569, ing the expression of a polynucleotide include but are not 834; 5,824,877: 5,563,04455 and 5,968,830); pineapple limited to Northern analysis, RT-PCR and dot-blot analysis (U.S. Pat. No. 5,952,543); poplar (U.S. Pat. No. 4,795,855); (Sambrook et al., Molecular Cloning: A Laboratory Manual, monocots in general (U.S. Pat. Nos. 5,591,616 and 6,037. 2nd Ed. Cold Spring Harbor Press, 1987). Polynucleotides or US 2011/O 191902 A1 Aug. 4, 2011 portions of the polynucleotides of the invention are thus use be identified. Two or more generations may be grown to ful as probes or primers, as herein defined, in methods for the ensure that the Subject phenotypic characteristics are stably identification of plants with altered levels of phloretinglyco maintained and inherited. Plants resulting from Such standard syltransferase or phlorizin. The polynucleotides of the inven breeding approaches also forman aspect of the present inven tion, or disclosed herein, may be used as probes in hybridiza tion. tion experiments, or as primers in PCR based experiments, designed to identify Such plants. BRIEF DESCRIPTION OF THE DRAWINGS 0272 Alternatively antibodies may be raised against (0277. The present invention will be better understood with polypeptides of the invention, or used in the methods of the reference to the accompanying drawings in which are invention. Methods for raising and using antibodies are stan described as follows: dard in the art (see for example: Antibodies, A Laboratory (0278 FIG. 1: Selection of MdPG1 as a key gene in ripen Manual, Harlow A Lane, Eds, Cold Spring Harbour Labora ing apple fruit tory, 1998). Such antibodies may be used in methods to detect 0279 A) The Venn diagram shows ESTs that significantly altered expression of polypeptides which modulate flower changed in expression using microarray analysis of RNA size in plants. Such methods may include ELISA (Kemeny, derived from three different treatments as described below: 1991, A Practical Guide to ELISA, NY Pergamon Press) and 0280 Fruit development: 8 time points from anthesis to Western analysis (Towbin & Gordon, 1994, JImmunol Meth ripe fruit, 0, 14, 25, 35, 60, 87, 132 and 146 days after full ods, 72,313). bloom. Ethylene induction: Ethylene knockout mutant apples 0273. These approaches for analysis of polynucleotide or induced to ripen with ethylene, and harvested 0, 4, 18, 96 and polypeptide expression and the selection of plants with 192 hours after ethylene induction. A 192 hour control (C) altered post-harvest storage characteristics are useful in con was also measured. Storage induction: Three different culti ventional breeding programs designed to produce varieties vars of apples were compared to each other before storage with altered post-harvest storage characteristics. (Cultivar comparisons) and after storage at 0°C. for 4 weeks. 0281. The expression profiles of the 143 cell wall related Plants genes were assessed in 3 unrelated microarray experiments that covered periods of physiological fruit softening: 0274 The term “plant' is intended to include a whole Microarray experiments were performed and analysed as plant, any part of a plant, a seed, a fruit, propagules and described in Schaffer et al (2007). progeny of a plant. 0282 B) Relative expression pattern of MdPG1 measured 0275. The term propagule' means any part of a plant that using qPCR may be used in reproduction or propagation, either sexual or 0283 RNA samples were extracted from ACC oxidase asexual, including seeds and cuttings. mutant apples to measure the effects of ethylene induced 0276. The plants of the invention may be grown and either softening on PG expression with or without a cold storage self-ed or crossed with a different plant strain and the result treatment. Timepoints analysed by qPCR are shown in the ing hybrids, with the desired phenotypic characteristics, may schematic below
nt -- 96 h -- 192 h (control)
Ol
N 4h -> 96 h -> 192 h (ethylene) Tree harvest ethylene N u1 nt -- 96 h -o- 192 h (cold control) 4 weeks Oh
N 4h -> 96 h -- 192 h (cold ethylene) ethylene
nt = not tested US 2011/O 191902 A1 Aug. 4, 2011
0284 cDNA was synthesized from 2 ug of total RNA in a (0300 FIG. 7: Western analysis of PG protein levels in six total volume of 50 mL with Superscript III reverse tran selected transgenic Royal Gala apple fruit Scriptase according to the manufacturer's instructions (Invit 0301 Protein samples were extracted and analysed as rogen). Controls with no Superscript III reverse transcriptase described in FIG. 3. were used to assess for potential genomic DNA contamina 0302 H=fruit at harvest tion. cDNA used for real-time RT-PCR was synthesized in (0303 2 w fruit after 2 weeks storage at 5° C. triplicate and optical density was measured for each sample. (0304) 16 w=fruit after 16 weeks storage at 5° C. qPCR reactions and normalization was performed as 0305 L=ladder, Precision Plus Protein Dual Colour Stan described in Schaffer et al (1998). Results show the strong dards (Bio-Rad) induction of MdPG1 expression by ethylene treatment after 0306 FIG. 8: Rate of water loss in transgenic vs control 96 hand 192 handby cold--ethylene treatment at 96 hand 192 fruit h. MdPG1 expression is induced by cold treatment alone but more slowly (after 192 h). 0307 A Rate of water loss from PG knockout and Royal 0285 FIG. 2: Firmness in transgenic PG knockout plants Gala fruit after 16 weeks storage at 5° C. followed by 5 weeks vs Royal Gala controls storage at room temperature. Water loss is expressed as grams 0286 Fruit firmness was measured destructively on indi of weight lost per day perg fresh weight of the fruit. Each bar vidual fruit (n=6-10 fruit per timepoint) at harvest, after 2 represents an individual fruit. weeks storage at room temperature (-20° C.) and after 16 0308 Conclusion: Compared to control RG (Royal Gala) weeks storage at 5° C. fruit, the two lines in which PG has been down-regulated most 0287 Firmness was measured using a puncture test strongly (lines PG41 and PG275) show the lowest rate of according to standard industry practise (Blanpied et al., water loss. 1978). This involved the localised removal of skin from two 0309 B Comparison of water loss from Royal Galacon opposing locations on the fruit equator, and recording the trol fruit and fruit in which the apple ACC oxidase gene has maximum force while driving a 7.9 mm cylindrical probe into been knocked out (see Schaffer et al 1998 for description of the outer cortex to a constant depth (8 mm) at a fixed speed (4 this line). Nb this fruit was not cold stored suggesting that mm/s) The puncture test and data capture was performed cold storing the apples may further accelerate water loss. using a Stable Micro Systems TA-XT plus Texture Analyser 0310 FIG.9: Reduced wrinkling in transgenic Royal Gala (Hertog et al 2001). fruit down-regulated for PG compared to Royal Gala control (0288 A) Fruit firmness in 10 knockout lines vs RG fruit (Royal Gala) control. 0311. Fruit were stored for 16 weeks at 5° C. then trans 0289 B) Fruit firmness measurement expressed as a % ferred to room temperature for 5 weeks at room temperature. offirmness at harvest. After this period transgenic fruit were substantially less 0290 SPI-Starch pattern index measurements were made wrinkled compared to controls which correlates with the against commercial standards. reduced loss of water from these fruit. 0291 FIG. 3: Western analysis of PG protein levels in 0312 FIG. 10: Toluidine blue stained sections of apple transgenic Royal Gala apple fruit at harvest from Royal Gala control and PG41 PG knockout line 0292 Fruit tissues were ground to a powder with mortar 0313 Apple fruit cortex sections were fixed in glutaralde and pestle under liquid nitrogen. Protein was extracted as hyde and embedded in LR-white resin. Thin sections of 1 uM described in Langenkapmperetal (1998). Proteins were sepa were stained with toluidine blue (0.1%). Sections were pre rated on 12% (w/v) SDS-Tris-Tricine gels using a Mini pared from control Royal Gala fruit and the PG41 PG knock PROTEAN3 electrophoresis system (Bio-Rad, Hercules, outline stored for 16 weeks at 5°C. Arrows indicate points of Calif., USA). Protein concentrations in each sample were pectin adhesion. measured using the QuBit Ouantitation System (Invitrogen) 0314 NB in control fruit the adhesion is reduced whilst in and verified on gels by Coomassie staining A polyclonal the PG41 lines the adhesion is maintained. This difference antibody raised to apple polygalacturonase was used to will have an effect on fruit softening and texture. immunolocalise PG protein in transgenic and control Royal 0315 FIG. 11: Immunolocalisation of non-esterified pec Gala apple plants. tin from Royal Gala control and PG41 PG knockout line 0293 Ladder-Precision Plus Protein Dual Colour Stan 0316 Fixation of apple fruit tissues and immunolocalisa dards (Bio-Rad), sizing is in kDa. tion using JIM5 antibodies was performed as described in 0294 FIG. 4: Western analysis of PG protein levels in Atkinson et al. (2002). Sections were prepared from control transgenic Royal Gala apple fruit after 2 weeks storage at Royal Gala fruit and the PG41 PG knockout line stored for 16 room temperature (-20°C.) weeks at 5°C. Magnification=x40. Arrows indicate points of 0295 Protein samples were extracted and analysed as JIM5 fluorescence. described in FIG. 3. 0317 NB in control fruit the fluorescence is reduced 0296 FIG. 5: Western analysis of PG protein levels in whilst in the PG41 lines the fluorescence is stronger. This transgenic Royal Gala apple fruit after 4 weeks storage at 5° result Suggests that more demethylated homogalacturonan is C. present at the junction points between cells in PG41 fruit vs 0297 Protein samples were extracted and analysed as control fruit. This difference will have an effect on fruit soft described in FIG. 3. ening and texture. 0298 FIG. 6: Western analysis of PG protein levels in 0318 FIG. 12: Tensile pull apart strength (upper panel) transgenic Royal Gala apple fruit after 16 weeks storage at 5° and flesh firmness measured with an 8 mm probe (lower C. panel) for fruit of PG41 lines versus control (Royal Gala) 0299 Protein samples were extracted and analysed as lines at harvest (yellow bars) and after 10 weeks storage at described in FIG. 3. 0.5°C. (magenta bars). US 2011/O 191902 A1 Aug. 4, 2011
0319 FIG. 13: Amino acid sequence of MDPG1 high 0325 Apple (Malus domestica Borkh. cv Royal Gala) rip lighting conserved polygalacturonase domains ens very differently than tomato and many other fruits (Redg 0320 FIG. 13 shows the position of four conserved well et al. 2008a; 2008b), because cell wall swelling is not one domains (I to IV) that are present in the plant and fungal of the cell wall modifications occurring during apple ripening sequences (Torki et al. 2000 (incorporated herein by refer ence)). The carboxylate group in the three aspartic acids in (Redgwell et al., 1997). There is minimal change in viscosity NTD and DD structures (domains I and II, respectively) may of cell walls, and minimal pectin Solubilization or degrada be a component of the catalytic site and the histidine residue tion during fruit ripening. This implies that any endo-PG in domain III is thought to participate to the catalytic reaction. isolated from ripening fruit of apple may have different char The well-conserved positively charged domain IV (RIK) con acteristics to endo-PGs isolated from ripening tomato fruit. In stitutes a likely candidate for ionic interactions with carboxy a range of apple cultivars there is a suggestion that levels of late groups present in the Substrate. polygalacturonase correlate with fruit firmness irrespective of ethylene production rate (Wakasa 2006). EXAMPLES 0321. The invention will now be illustrated with reference Example 2 to the following non-limiting examples. Production of Plants with Reduced Expression of Example 1 MdPG1 Selection of MdPG1 as a Candidate Gene for Alter ing Post-Harvest Storage Life in Apple Fruit 0326 Ten transgenic Royal Gala lines were created con 0322 Three microarray experiments that measured apples taining MdPG1 expressed in an antisense orientation driven that underwent ripening were analysed. These included an by a strong constitutive promoter (35S promoter). The fruit ethylene induced ripening series (Schaffer et al 2007), a fruit specific polygalacturonase cDNA clone MdBG1 (formerly development series (Janssen et al 2008) and a cold storage GDPG1, Atkinson 1994), was cloned into pART7 as treatment (manuscript in preparation). 290 cell wall related described previously (Atkinson et al. 2002). A clone with the genes were identified by homology screening in the HortRe PG gene in the antisense orientation was digested with NotI search Apple EST collection. Of these, 10 increased in and cloned into the binary vector p ART27. The binary was expression late in fruit development, 9 increased in expres electroporated into Agrobacterium tumefaciens strain sion upon the addition of exogenous ethylene, and 10 LBA4404. Transgenic apple Royal Gala shoots were pro increased in expression during 2 and a half months of cold duced using the method of Yao et al. (1995) and maintained in storage. Of these genes, three were found to be in common to a containment greenhouse under identical conditions (ambi all treatments. Of the three, MdPG1 showed the greatest ent light and temperature) to wild-type plants. Plants were change in expression. Further analysis of this gene in trans transferred to chillers for 8-10 weeks each year to meet winter genic apple lines down-regulated for the Md.ACO gene chilling requirements. Flowers were hand-pollinated each showed that MdBG1 is up-regulated in an ethylene dependent spring and fruit harvested in autumn when aroma Volatiles and cold dependent ripening manner (FIG. 1). could be detected. 0323 Although reduction of expression of polygalactur onases (PGs) has been proposed as an approach to improving storage characteristics in fruit, Success has been limited. This Example 3 may be partly due to the large number of PGs that appear to be present in plants. For example Arabidopsis alone has been Fruit of Plants Produced by the Methods of the reported to contain approximately 52 different PG genes. Invention Show Reduced Softening During Post 0324 Transgenic plants have been used to study the role of Harvest Storage endo-PGs in vivo. In tomato (Lycopersicon esculentum), down-regulation of the fruit-specific PG gene pTOM6 under 0327 Fivelines, of the 10 described in Example 2, showed the control of the constitutive cauliflower mosaic virus 35S less softening than the wild type control after two weeks at promoter showed reduced depolymerization of pectin poly room temperature (FIG. 2), and 2 lines (PG275 and PG41) mers in fruit (Smith et al., 1990). Overexpression of PG in the showed significantly less softening after 16 weeks at 5° C. ripening inhibited mutant rin background restored PG activ storage. This correlated with previous experiments where ity and pectin degradation in fruit (Giovannoni et al., 1989). apples from the line PG41 showed much reduced softening In both cases, only the fruit was affected by the transgene compared to the control apples. The decreased softening in expression; therefore, the gene product isolated from tomato this line has been shown for fruit collected over 3 growing fruit appeared to have fruit specific PG activity. Further experiments where the pTOM6 gene was overexpressed in seasons (Table 1). tobacco (Nicotiana tabacum; Osteryoung et al., 1990) 0328. Firmness was measured using a puncture test showed that the tomato protein was properly processed and according to standard industry practise (Blanpied et al., localized in the cell walls of leaves in tobacco. The enzyme 1978). This involved the localised removal of skin from two showed activity when extracted from transgenic tobacco opposing locations on the fruit equator, and recording the leaves and tested against tobacco cell wall extracts in vitro. maximum force while driving a 7.9 mm cylindrical probe into However, no changes in leaf phenotype were observed, nor the outer cortex to a constant depth (8 mm) at a fixed speed (4 were there any alterations to the pectins in the tobacco cell mm/s) The puncture test and data capture was performed walls in vivo. Expressing and given PG gene in plants thee using a Stable Micro Systems TA-XT plus Texture Analyser fore may give unpredictable results. (Hertogetal 2001). US 2011/O 191902 A1 Aug. 4, 2011
TABLE 1. Firmness in PG41 vs control apples over 3 years 2005 Storage 2007 Storage 2008 Storage Harvest 30 wks, 5° C. Harvest 32 wks, 5° C. Harvest 16 wks, 5° C. Probe 11 11 11 11 8.5 8.5 (mm) firmness firmness firmness firmness firmness firmness PGA41 no data 6.18 9.25 7.0 S.O6 3.SO control no data 2.83 4.7 4.0 4.64 2.49
0329. Firmer fruit is a desirable characteristic as sensory/ (0.00273 and 0.00237 g/day/g FW respectively) compared to consumer trials show that consumers prefer firmer fruit. the untransformed control (0.0046 g/day/g FW). The other transgenic lines showed a range between 0.00299 and Example 4 0.00317 g/day/g FW) (FIG. 8). These numbers were much Levels of MdPG1 Protein Correlate with Rate of larger than those found in a separate water loss experiment Softening with a non-ripening mutant ACO antisense apples and Royal Gala lines that had not been cold stored (FIG. 8), suggesting 0330. The mature ORF of MdPG1 was amplified by PCR that cold storing the apples may further accelerate water loss. using primers RA136 5'-ACGGGATCCG CTCCGGCCAA Apples from the PG41 and PG275 lines also showed less AACCATTAGC-3' and RA137 5'-ATAGTTTA shriveling compared at 5 weeks RT after transfer from 16 GCGGCCGCTTAA CATCTAGGGG AGACAAC-3'. The weeks cold storage (FIG. 9). insert was excised with BamHI and NotI (underlined in the primers) and ligated into corresponding sites of the pET-30a Example 6 (+) vector (Novagen, Madison, Wis., USA). pETMdPG1 was Fruit of Plants Produced by the Methods of the transformed into BL21 cells containing the pyss plasmid Invention Show Increased Juiciness During Post and recombinant His-tagged protein purified by Ni-affinity Harvest Storage chromatography under denaturing conditions (Schröder et al. 1998). Purified recombinant forms of MdPG1 protein cut Microscopic Analysis of PG41 Lines and Untransformed from a polyacrylamide gel was used to raise a polyclonal Controls antibody in rabbits. 0333 Sectioning cells from Royal Gala apples following a 0331 Levels of MidPG1 protein were measured on west 16 week cold storage period revealed that the cell-to-cell ern blots using polyclonal antibodies raised to the mature adhesion was significantly weakened (with presumably pec MdPG1 protein. For each transgenic line, described in tin junctions between cells showing clear regions that have Example 3 above, protein was extracted from apples at har pulled apart FIG. 10A). Sections of cells in the PG41 lines Vest, after two weeks room temperature storage and after 16 show no pulling apart (FIG. 10B). Additionally antibody weeks cold storage. Atharvest no MdPG1 was detected in any staining of the PG41 lines showed a maintenance of the of the apples except line PG290 (FIG. 3), After 2 weeks demethylated homogalacturans in cell corners (FIG. 11) com storage at room temperature (RT) it was found that both the pared to the Royal Gala control, that are targeted by PG Royal Gala lines and PG290 showed a significant level of (identified using a JIM5 antibody). This suggests that PG. Lines PG7, PG8, PG 17, and PG 164 had a detectable level decreasing the level of PG reduces cells breaking between the of PG(FIG. 4). After 4 weeks of cold storage lines PG7, PG8, cell boundaries rather than across the cells. It has been pro PG30 and PG164 had a detectable level of PG (lines 213B, posed that that the difference between juicy apples and mealy PG275 and PG290 were not assayed) (FIG. 5). At 16 weeks apples is due to the way that the cells are disrupted during a storage lines except PG41 and PG275 showed significant bite action. Hallett et al have shown that juicyness is not a levels of PG (FIG. 6). Comparison across time points there measure of water content rather as mealy and juicy apples was a strong correlation of levels of PG and rate of softening. contain the same amount of water. It has been suggested that Lines PG30 and PG40 showed little softening at 2 weeks RT juicy apples break across cells releasing the juice while mealy and showed very low levels of PG at this time point. PG41 apples break between cells giving a much dryer mouth feel. showed no detectable PG and PG275 showed very low levels The loss of cell to cell adhesion in the control lines Suggest of PG both of which were the firmest apples after 16 weeks that the apples would have a more mealy texture, and the storage (FIG. 7). PG41 apples would be more juicy (this cannot be confirmed due to restrictions on eating transgenic apples in this country). Example 5 From these results it is anticipated that the PG knockout apples would also be crisper and crunchier than the Royal Fruit of Plants Produced by the Methods of the gala controls. When cutting the apples after storage they Invention Show Reduced Water Loss During Post appeared to maintain their crispness. Harvest Storage Example 7 0332 Apples from 8 independent transformant lines, along with the control Royal Gala apples were left at room Fruit of Plants Produced by the Methods of the temperature for 1 month following a 16 week storage period Invention Show Altered Wax Composition at 4 degrees, and weighed every two weeks. It was found that 0334 PG antisense lines PG 17 and PG275 both had a the lines PG41 and PG275 showed a lower rate of water loss waxy feel compared to the Royal Gala control providing US 2011/O 191902 A1 Aug. 4, 2011
evidence that altered expression in the method of the inven 0339. Two variant sequences were identified as sum tion can result in altered wax production. marised in the table below. Example 8 Fruit of Plants Produced by the Methods of the Maius Polynucleotide Polypeptide Invention Show Reduced Post-Harvest Storage Rots/ Polygalacturonase species SEQ ID NO: SEQID NO: Infections MSPG1 Sieboidii 6 2 0335 Control fruit were subject to infection by posthar MSPG2 Sieboidii 7 3 Vest pathogens after long term storage at 5° C. In contrast PG41 lines rarely showed infection. This effect may be due to (0340. The table below shows the % identity between the a reduction in microcracks on the surface of the fruit which MdPG1 and variant polypeptide sequences. provide an entry point for pathogen invasion. Fruit Storage at 5° C. 0336 Commercial fruit storage is carried out at 1° C. in McPG1 MSPG1 MSPG2 controlled/modified atmosphere conditions. PG41 apples in McPG1 100% 92.3 95.2 this study were stored at less than optimal conditions and still MSPG1 100% No overlap maintained fruit quality. This may allow fruit to stored at MSPG2 100% slightly higher temperatures thereby reducing costs. 0341 The function of these variants can be confirmed Example 9 using the methods described in the examples above. Fruit of Plants Produced by the Methods of the Example 11 Invention Show Mostly Normal Ripening Attributes Fruit of Plants Produced by the Methods of the PG41 Apples Show Normal Ripening Attributes Invention Show Increased Tensile Strength and Firm 0337 To assess whether any other ripening attribute was ness in Commercial Storage Conditions altered in the PG41 mutant that may contribute to the pheno 0342. 30 fruit from the PG41 lines which had no detect type, internal ethylenes, starch pattern index (SPI) and able fruit ripening endopolygalacturonase 1 were harvested soluble solids content (SSC) were measured at harvest, and along with 30 untransformed Royal Gala (RG) controls. 15 ethylene was measured 16 weeks after cold storage. From the fruit had 1 mm skin removed in 4 quadrants in the equatorial SPI the Royal Gala apples appeared to be slightly more region of the apple and were measured at harvest from each mature than the PG knock out lines at harvest, but after 16 line for puncture firmness was measured (with 4 different weeks cold storage the PG41 lines and Royal Gala controls probe sizes) (Tablela) using a TAXT texture analyzer (Stable were producing similar amounts of ethylene (FIG. 12), Sug Micro Systems, Ltd, UK) as described in Johnston et al gesting that the reduced softening is not due to decreased (2009). Cores from the cortex tissue were taken and tensile levels of ethylene. strength of these were measured using the TAXT texture analyzer Example 10 (0343 15 apples from each line were stored at 0.5°C. for 10 Identification of Variants of the MidPG1 weeks under commercial storage conditions. Following this time apples were tested again for tensile strength and flesh 0338. The MidPG1 sequence was used to identify ortholo puncture firmness. Additionally these samples were also gous PG genes from HortResearch proprietary sequence assessed for amount of juice released by a commercial juicer databases. to assess levels of juiciness. TABLE 1. Results oftensile strength and Firmness Storage Absolute values % change during storage
Data time (wks) Control PG41 Control PG41 Average of Tens Max O 13.11.0 12.5 O.6 5140 34.26 Force (N) 10 6.3 + O.7 8.2 + 1.0 Average of Force 11 mm O 84.4 2.2 78.6 3.3 30.94 16.91 Probe (N) 10 58.314 65.3 2.4 Average of Force 8 mm O 45.8 1.5 44.3 2.0 33.08 23.70 Probe (N) 10 30.6+ O.6 33.81.0 Average of Force 5 mm O 18.7+ O.6 18.9 O.8 28.37 22.73 Probe (N) 10 13.4 O.3 14.6 0.5 Average of Force 2 mm O 4.1 + O.2 4.2 + O.2 34.97 28.20 Probe (N) 10 2.7 - O 3.0 C) US 2011/O 191902 A1 Aug. 4, 2011 20
0344. There were no clear differences between the control 0355 Hamilton, A. J., Lycett, G. W. Grierson, D. 1990. RG lines and the PG 41 lines at harvest. But after 10 weeks Antisense Gene That Inhibits Synthesis of the Hormone storage there was a significant increase in both tensile Ethylene in Transgenic Plants. Nature 346, 284-287. strength and firmness. The 7 N increase (65.3 N-58.3) in 0356. Harker, F. R., Maindonald, J., Murray, S. H., Gun firmness of PG41 apples relative to RG control apples mea son, F. A., Hallett, I. C. and Walker, S. B. 2002. Sensory Sured with the 11 mm probe following storage is larger than interpretation of instrumental measurements 1: texture of the minimum 6 N difference that a trained sensory panel can apple fruit. Postharvest Biology and Technology, 24, 225 detect (Harker etal 2002) strongly indicating that in a sensory 239. trial the PG41 fruit would be scored as better textured than the 0357 Hertog MLATM, Nicholson SE, Banks N. 2001. RG control after storage (FIG. 12). When the original firm The effect of modified atmospheres on the rate offirmness ness of the PG41 fruit is taken into account then there is only changes in Braeburn apples. Postharvest Biology and a 16% loss offirmness compared to a 31% loss offirmness in Technology, 23, 175-184. the control fruit. 0358 Janssen, B.J., Thodey, K., Schaffer, R. J., Alba, R., Balakrishnan, L., Bishop, R., Bowen, J. H., Crowhurst, R. REFERENCES N., Gleave, A. P. Ledger, S., McArtney, S. Pichler, F. B., Snowden, K. C., Ward, S. 2008. Global gene expression (0345 Atkinson, R. G. 1994. A cDNA clone for endopo analysis of apple fruit development from the floral bud to lygalacturonase gene from apple. Plant Physiol. 195, 1437 ripe fruit. BMC Plant Biol 8, 16. 1438. 0359 Johnston J. W. Gunaseelan K, Pidakala P. Wang M, 0346 Atkinson, R. G., Schröder, R., Hallett, I. C., Cohen, Schaffer R.J. (2009) Coordination of early and late ripen D., MacRae, E. A. 2002. Overexpression of polygalactur ing events in apples is regulated through differential sen onase in transgenic apple trees leads to a range of novel sitivities to ethylene. J. Exp. Bot 60,2689-2699 phenotypes involving changes in cell adhesion. Plant 0360 Langenkamper, G. McHale, R., Gardner, R. C., Physiol. 129, 122-133. MacRae, E. 1998. Sucrose-phosphate synthase steady 0347 Ayub, R., Guis, M., Ben Amor, M., Gillot, L., state mRNA increases in ripening kiwifruit. Plant Mol. Roustan, J. P. Latche, A., Bouzayen, M., Pech, J. C. 1996. Biol. 36, 857-69. Expression of ACC oxidase antisense gene inhibits ripen 0361 Langley, K. R., Martin, A., Stenning, R., Murray, A. ing of cantaloupe melon fruits. Nat. Biotechnol. 14, 862-6. J., Hobson, G. E., Schuch, W. W., Bird, C. R. 1994. 0348 Blanpied G D, Bramlage W J, Dewey D. H. LaBelle Mechanical and Optical Assessment of the Ripening of RL, Massey L. M. Mattus G. E. Stiles W. C. Watada A. E. Tomato Fruit with Reduced Polygalacturonase Activity.J. 1978. A standardized method for collecting apple pressure Sci. Food Agric. 66, 547-554. test data. New York's Food and Life Sciences Bulletin, 74 0362. Oeller, P. W., Lu, M. W., Taylor, L. P., Pike, D. A., (August), 3-8. Theologis, A. 1991. Reversible inhibition of tomato fruit senescence by antisense RNA. Science 254, 437-9. (0349 Brummell, D. A., Harpster, M. H., Civello, P. M., 0363 Osteryoung, K. W., Toenjes, K., Hall, B., Winkler, Palys, J. M., Bennett, A. B., Dunsmuir, P. 1999. Modifica V., Bennett, A. B. 1990. Analysis of tomato polygalactur tion of expansin protein abundance in tomato fruit alters onase expression in transgenic tobacco. Plant Cell 2, 1239 Softening and cell wall polymer metabolism during ripen 48. ing. Plant Cell 11, 2203-16. 0364 Powell, A. L., Kalamaki, M. S. Kurien, P.A., Gur 0350 Cantu, D., Vicente, A. R., Greve, L. C., Dewey, F. rieri, S., Bennett, A. B. 2003. Simultaneous transgenic M., Bennett, A. B., Labavitch, J. M., Powell, A. L. 2008. suppression of LePG and LeExpl influences fruit texture The intersection between cell wall disassembly, ripening, and juice viscosity in a fresh market tomato variety. J. and fruit susceptibility to Botrytis cinerea. Proc. Natl. Agric. Food Chem. 51, 7450-5. Acad. Sci. U.S.A. 105,859-64. 0365 Schröder, R., Atkinson, R. G., Langenkämper, G., 0351 Carey, A.T., Smith, D. L., Harrison, E., Bird, C. R., Redgwell, R. J. 1998. Biochemical and molecular charac Gross, K. C., Seymour, G. B., Tucker, G. A. 2001. Down terisation of Xyloglucan endotransglycosylase from ripe regulation of a ripening-related beta-galactosidase gene kiwifruit. Planta 204, 242-251. (TBG1) in transgenic tomato fruits. J. Exp. Bot. 52, 663 0366 Redgwell, R. J., Curti, D., Gehin-Delval, C. 2008a. 668. Physicochemical properties of cell wall materials from 0352 Carrington, C., Greve, L. C., Labavitch, J. M. 1993. apple, kiwifruit and tomato. European Food Research and Cell Wall Metabolism in Ripening Fruit (VI. Effect of the Technology 227,607-618. Antisense Polygalacturonase Gene on Cell Wall Changes 0367 Redgwell, R. J., Curti, D., Gehin-Delval, C. 2008b. Accompanying Ripening in Transgenic Tomatoes). Plant Role of pectic polysaccharides in structural integrity of Physiol. 103,429-434. apple cell wall material. European Food Research and 0353 Giovannoni, J. J., DellaPenna, D., Bennett, A. B., Technology 227, 1025-1033. Fischer, R. L. 1989. Expression of a chimeric polygalac 0368 Redgwell, R.J., Fischer, M., Kendal, E., MacRae, E. turonase gene in transgenic rin (ripening inhibitor) tomato A. 1997. Galactose loss and fruit ripening: high-molecular fruit results in polyuronide degradation but not fruit soft weight arabinogalactans in the pectic polysaccharides of ening. Plant Cell 1, 53-63. fruit cell walls. Planta 203, 174-181. 0354 Hellens, R. P. Allan, A. C., Friel, E.N., Bolitho, K., 0369 Saladie, M., Rose, J. K. C., Cosgrove, D.J., Catalá, Grafton, K., Templeton, M.D., Karunairetnam, S., Gleave, C. 2006. Characterization of a new xyloglucan endotrans A. P. and Laing, W. A. (2005) Transient expression vectors glucosylase/hydrolase (XTH) from ripening tomato fruit for functional genomics, quantification of promoter activ and implications for the diverse modes of enzymic action. ity and RNA silencing in plants. Plant Methods 1, 13 Plant J. 47, 282-295. US 2011/O 191902 A1 Aug. 4, 2011 21
0370 Santiago-Domenech, N., Jimenez-Bemudez, S., beta-1,4-glucanase gene together with the Erwinia pectate Matas, A.J., Rose, J. K., Munoz-Blanco, J., Mercado, J.A., lyase and polygalacturonase genes in Saccharomyces cer Quesada, M. A. 2008. Antisense inhibition of a pectate evisiae. Curr Genet. 27(1):17-22. lyase gene Supports a role for pectin depolymerization in strawberry fruit softening. J. Exp. Bot. 59,2769-79. 0371 Schaffer, R. J., Friel, E. N., Souleyre, E.J., Bolitho, SUMMARY OF SEQUENCES K., Thodey, K. Ledger, S., Bowen, J. H. Ma, J. H., Nain, B., Cohen, D., Gleave, A. P., Crowhurst, R.N., Janssen, B. 0378 J.Yao, J. L., Newcomb, R. D. 2007. A genomics approach reveals that aroma production in apple is controlled by ethylene predominantly at the final step in each biosyn thetic pathway. Plant Physiol. 144, 1899-912. SEQID NO: Type Species Reference 0372 Schuch, W., Kanczler, J., Robertson, D., Hobson, 1 polypeptide Maius X domestica Md PG1, G., Tucker, G., Grierson, D., Bright, S., Bird, C. 1991. polygalacturonase, Fruit-Quality Characteristics of Transgenic Tomato Fruit Genbank accession with Altered Polygalacturonase Activity. HortScience 26, number L27743 1517-1520. polypeptide Maius Sieboidii MsPG1, 0373) Smith, C. J. Watson, C. F., Bird, C. R., Ray, J., polygalacturonase Schuch, W. Grierson, D. 1990. Expression of a truncated polypeptide Maius Sieboidii MsPG2, tomato polygalacturonase gene inhibits expression of the polygalacturonase endogenous gene in transgenic plants. Mol. Gen. Genet. polynucleotide Maius X domestica, Md PG1, 224, 477-81. polygalacturonase 0374 Torki, M., Mandaron, P., Mache, R., and Falconet, promoter sequence D. 2000. Characterization of a ubiquitous expressed gene and part of first family encoding polygalacturonase in Arabidopsis exon. Genbank accession number thaliana. Gene. 2000 Jan. 25: 242(1-2):427-36. AFO31233) 0375 Wakasa, Y., Kudo, H., Ishikawa, R., Akada, S., polynucleotide Maius X domestica, Md PG1, Senda, M., Niizeki, M., Harada, T. 2006. Low expression polygalacturonase, of an endopolygalacturonase gene in apple fruit with long cDNA term storage potential. Postharvest Biol. Technol.39, 193 polynucleotide Maius Sieboidii MsPG1, 198. polygalacturonase, 0376 Yao, J. L., Cohen, D., Atkinson, R., Richardson, K., cDNA Morris, B. 1995. Regeneration of transgenic plants from polynucleotide Maius Sieboidii MsPG2, the commercial apple cultivar Royal Gala. Plant Cell polygalacturonase, Reports 14, 407-412. cDNA 0377 van Rensburg, P. van Zyl. W. H., Pretorius I. S. 1994. Expression of the Butyrivibrio fibrisolvens endo
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 7
<21 Oc SEO ID NO 1 <211 LENGTH: 460 <212> TYPE PRT <213> ORGANISM: Malus x domestica
<4 OOs SEQUENCE: 1
Met Ala Lieu Lys Thr Glin Lieu Lleu Trp Ser Phe Wall Wa. l Wall Phe Wall 1. 5 15
Wall Ser Phe Ser Th Thr Ser Ser Gly Ser Ser Phe Glin Glu Wall 25 3 O
ASn Ala Lieu. His Ser Tyr Wall Asp His Wall Asp Asp Lys Glu Ser Gly 35 4 O 45
Asn. Ser Arg Ala Tyr Pro Ser Thir Asp Thir Il e Glu Gly Lell SO 55 60
Wal Met Glu Luell Ile Arg Pro Arg Thir Glin Luell Phe Ser Ser Arg 70
Lieu. Asn Thir Ile Thir Gly Gly Ile Ala Thir Ser Se r Ala Pro Ala 85 90 95 US 2011/O 191902 A1 Aug. 4, 2011 22
- Continued
Llys Thir Ile Ser Val Asp Asp Phe Gly Ala Lys Gly Asn Gly Ala Asp 1OO 105 11 O Asp Thr Glin Ala Phe Wall Lys Ala Trp Lys Ala Ala Cys Ser Ser Ser 115 12 O 125 Gly Ala Met Val Lieu Val Val Pro Gln Lys Asn Tyr Lieu Val Arg Pro 13 O 135 14 O Ile Glu Phe Ser Gly Pro Cys Llys Ser Glin Lieu. Thir Lieu. Glin Ile Tyr 145 150 155 160 Gly. Thir Ile Glu Ala Ser Glu Asp Arg Ser Ile Tyr Lys Asp Ile Asp 1.65 17O 17s His Trp Lieu. Ile Phe Asp Asn Val Glin Asn Lieu. Lieu Val Val Gly Pro 18O 185 19 O Gly. Thir Ile Asin Gly Asn Gly Asn. Ile Trp Trp Lys Asn. Ser Cys Llys 195 2OO 2O5 Ile Llys Pro Gln Pro Pro Cys Gly Thr Tyr Ala Pro Thr Ala Val Thr 21 O 215 22O Phe Asn Arg Cys Asn. Asn Lieu Val Val Lys Asn Lieu. Asn. Ile Glin Asp 225 23 O 235 24 O Ala Glin Glin Ile His Val Ile Phe Glin Asn. Cys Ile Asin Val Glin Ala 245 250 255 Ser Cys Lieu. Thr Val Thr Ala Pro Glu Asp Ser Pro Asn Thr Asp Gly 26 O 265 27 O
Ile His Wall. Thir Asn. Thir Glin Asn. Ile Thir Ile Ser Ser Ser Wall Ile 27s 28O 285 Gly Thr Gly Asp Asp Cys Ile Ser Ile Val Ser Gly Ser Glin Arg Val 29 O 295 3 OO Glin Ala Thr Asp Ile Thr Cys Gly Pro Gly His Gly Ile Ser Ile Gly 3. OS 310 315 32O Ser Lieu. Gly Glu Asp Gly Ser Glu Asp His Val Ser Gly Val Phe Val 3.25 330 335 Asn Gly Ala Lys Lieu. Ser Gly. Thir Ser Asn Gly Lieu. Arg Ile Llys Thr 34 O 345 35. O Trp Lys Gly Gly Ser Gly Ser Ala Thr Asn Ile Val Phe Glin Asn Val 355 360 365 Gln Met Asn Asp Val Thr Asn Pro Ile Ile Ile Asp Glin Asn Tyr Cys 37 O 375 38O Asp His Llys Thir Lys Asp Cys Lys Glin Glin Llys Ser Ala Val Glin Val 385 390 395 4 OO Lys Asn Val Lieu. Tyr Glin Asn. Ile Arg Gly. Thir Ser Ala Ser Gly Asp 4 OS 41O 415 Ala Ile Thr Lieu. Asn Cys Ser Glin Ser Val Pro Cys Glin Gly Ile Val 42O 425 43 O Lieu. Glin Ser Val Glin Lieu. Glin Asn Gly Arg Ala Glu. Cys Asn. Asn. Wall 435 44 O 445 Gln Pro Ala Tyr Lys Gly Val Val Ser Pro Arg Cys 450 45.5 460
<210s, SEQ ID NO 2 &211s LENGTH: 247 212. TYPE: PRT <213> ORGANISM: Malus Sieboldii US 2011/O 191902 A1 Aug. 4, 2011 23
- Continued <4 OOs, SEQUENCE: 2 Met Ala Leu Lys Thr Glin Lieu. Leu Trp Ser Phe Val Val Val Phe Val 1. 5 1O 15 Val Ser Phe Ser Thr Thr Ser Cys Ser Gly Ser Ser Phe Glin Glu Val 2O 25 3O Asn Ala Lieu. His Ser Tyr Val Asp His Val Asp Asp Llys Glu Ser Gly 35 4 O 45 Cys Asn Ser Arg Thr Tyr Pro Ser Tyr Thr Asp Thr Ile Ile Glu Gly SO 55 6 O Lieu Lys Phe Met Glu Lieu. Ile Arg Pro Ile Thr Gln Leu Phe Ser Ser 65 70 7s 8O Arg Llys His Asn. Thir Ile Thr Gly Gly Ile Ala Thir Ser Ser Ala Arg 85 90 95 Ala Lys Thir Ile Ser Val Asp Asp Phe Gly Ala Lys Gly Asp Gly Ala 1OO 105 11 O Asp Asp Thr Glin Ala Phe Glu Lys Ala Trp Lys Ala Ala Cys Ser Ser 115 12 O 125 Ser Gly Ala Met Val Lieu Val Val Pro Gln Lys Asn Tyr Lieu Val Gly 13 O 135 14 O Pro Ile Glu Phe Ser Gly Pro Cys Llys Ser Arg Lieu. Thir Lieu. Glin Ile 145 150 155 160 Tyr Gly. Thir Ile Glu Ala Ser Glu Asp Arg Ser Ile Tyr Lys Asp Thr 1.65 17O 17s Asp His Trp Lieu. Ile Phe Asp Asn Val Glin Asn Lieu Val Val Val Gly 18O 185 19 O Pro Gly. Thir Ile Asn Gly Asn Gly Asn. Ile Trp Trp Lys Asn. Ser Cys 195 2OO 2O5 Lys Ile Llys Pro Gln Pro Pro Cys Asp Thr Tyr Ala Pro Met Ala Val 21 O 215 22O Thir Phe Asp Llys Cys Asn. Asn Lieu Val Val Lys Asn Lieu. Asn. Ile Arg 225 23 O 235 24 O Asp Ala Glin Glin Met His Val 245
<210s, SEQ ID NO 3 &211s LENGTH: 1.87 212. TYPE: PRT <213> ORGANISM: Malus Sieboldii
<4 OOs, SEQUENCE: 3 His Val Ser Asn Thr Glin Asn Ile Thr Ile Ser Ser Ser Val Ile Gly 1. 5 1O 15 Thr Gly Asp Asp Cys Ile Ser Ile Val Ser Gly Ser Glin Arg Val Glin 2O 25 3O Ala Thr Asp Ile Thr Cys Gly Pro Gly His Gly Ile Ser Ile Gly Ser 35 4 O 45 Lieu. Gly Glu Asp Gly Ser Lys Asp His Val Ser Gly Val Phe Val Asn SO 55 6 O Gly Ala Lys Lieu. Ser Gly. Thir Ser Asn Gly Lieu. Arg Ile Llys Thir Trp 65 70 7s 8O Glu Gly Gly Ser Gly Ser Ala Thr Asn Ile Val Phe Glin Asn Val Glin 85 90 95 US 2011/O 191902 A1 Aug. 4, 2011 24
- Continued Met Asn Asp Wall Ala Asn Pro Ile Ile Ile Asp Glin Asn Tyr Arg Asp 1OO 105 11 O His Llys Thir Lys Asp Cys Lys Glin Gln Lys Ser Ala Val Glin Val Lys 115 12 O 125 Asn Val Lieu. Tyr Lys Asn. Ile Arg Gly. Thir Ser Ala Ser Arg Asp Ala 13 O 135 14 O Ile Thr Lieu. Asn Cys Ser Pro Ser Val Pro Cys Glin Gly Ile Val Lieu. 145 150 155 160 Glin Ser Val Glin Lieu. Glin Asn Gly Arg Ala Glu. Cys Asn. Asn. Wall Lys 1.65 17O 17s Pro Ala Tyr Lys Gly Val Val Ser Pro Arg Cys 18O 185
<210s, SEQ ID NO 4 &211s LENGTH: 2839 &212s. TYPE: DNA <213> ORGANISM: Malus x domestica
<4 OOs, SEQUENCE: 4 gaattcaaaa aaactatttg gaccattccg agcaagttcta t ctaaatago: aatagcatca 6 O ctictat atta ttgcagttct tattalaga.gt gtgtataata ttgtacttgt atccaaaa.ca 12 O acaatcctgg c tatttittac at attcaata taaagttcca atacgat cac tdattgatta 18O attggitatica agtacaatac ttctgcatct citcttaaaaa acatcaacaa togcattacgt. 24 O gttgtagatt gttatcagag ticggc cctgg CCC agtgtca C cagggcgac cqcttgaggit 3OO c caaaaaaac gag toggcacc aaaaaaaaat taagttggitt catatatgta taaggittittg 360 ttittatt citt gatatatagt aaacgct cat attaaatago: aatatgttitt agatgacagg 42O gtggittitt at ttggtttitt.c cqtgcccacc taggtttaat tcc ct ct ct c aacataaagg 48O ggtag titt co cittctattitt att catgtct actittaaata acaaacaaac attaattagc 54 O ttattaaatg toggcatttitc agtag togtt ttttgttitt.c agtttgaatt aactic goaaa 6OO at catgttcaagtttgttcta aatataaatt taggttttitt at atttgttgataatcttitt 660 catggittaag taataaactt gtgat catta t citttittatt gaacgaagta ttgttgacaa 72 O tccaaaaata t catcc taca cittctttgta taattitttitc ctitatgattt tatatatttg 78O gaatgaac cq caacttitt ca tdagcgct cq aaaaacaaaa acatagttga attac tatgt 84 O gcc ctittggit agtagcaatt togttgtttitc atttgttgct tacatataga aattagagat 9 OO cittaaatatgaaaaattic at ttcaaaagtt toataatgtg caagtagctt gtagat cagt 96.O tagittaaaag togttcagott gttcact caat gactic gttitt caaattt cot caccg tattt O2O citgatgagtt tagtgtaaat tacccitat catttgttcaaaa aagt cataat gtgaaaaagg O8O tctattittct ttaatatt ca atgtacaa.ca tacaaatact aagataaata attatttitat 14 O gtgaagtt at gttagggcat atttittcaat gtc.gc.ctagg gcct cataaa act caggata 2OO ggcc ttgatt gttatatatg gtact aaa.ca aaagttt coa aaatacaaag tittaaaaaga 26 O ttcaagtgga attittgaaga attittaaaag tattataaat tittgata acc cct coagttg 32O attaattgcg ggggatgtgg tdatcatgac cqctatggta gttcatggag caacct actt 38O gttcc.cccac titatgattga cqcatttitta citg tatgaac at cataatca gaaccottcq 44 O ttct citttgt catcatcgaa agat catata tdcaaaaact cattaaattig agagatttitt SOO US 2011/O 191902 A1 Aug. 4, 2011 25
- Continued ttagcc attc atatgtatica tacaaataga cqgttcatca taaggatgct acttgttacc 1560 aaaaac attg attggtttga catatatgga tiggg taalaca atcto caaat ggaat attitt 162O ttittagatat gat attitcga tigatgataaa gagaatgaac gigg to aaata ataatgttca 168O tatggtgaag atgcgtaaat cataagtgaa gggacaagtt ggttc cctat ggaggacaca 1740 agcattatt c gtttittaggt g tatt caatt agaattgtaa atgaatctat aaaagttcag 18OO gaat attcag to agaatgtt aaacaagctt atagaacticc atacaaattic aggtg tatta 1860 atcaattaaa attittaaagg attittataaa agt caacaga aatctgagtg tatt caaaga 1920 agattittgaaaaagttctaag aaagttagag td tattgatc agtaataatt tdattittaaa 198O gaattittaaa atgatacatt ttagtaaatt tdaaggaatt toatagagta tittaa.ccctt 2O4. O aataaatcat acttctgtaa agt coattaa aaaactic cat caactitt cat aaattgaaac 21OO atttittaaat coataaaagt togaac cqaat c tattitt cat at attattitt atacatgcaa. 216 O cgacttacgt ttalacataa gggatgcaat gcatggcgca gaaagt cata gag toggcaa. 222 O agacat catt togtotgaat citct catgtc. cdagaac cca tacct caaga gcc caagacg 228O acacaataca caacaat cac cqt caatacc cittct ctitcc gctgcctata aataccalatg 234 O gaaatcc.cac gacattctica ccaaatcatc at cactitgaa cacac caatic cittacacttic 24 OO tagctacaat tctaagttt c catttitccaa catcc catca cattgttcaa aaatat catc 246 O agcctogagt tagggittt at tat cottcgt gaccctoctt ttagtatttg gttctttittg 252O aaagacgatt tottaggtgt ttctaggcct ctago catct totatict cac caaaaaaaaa 2580 tat attacca gctgtttaca ccaaattaaa tagtaaaaag aaa.gcatcaa tdgctittaaa 264 O aacacagttgttgttggtcat ttgttgttgt ttttgttgtt to citt cagta caactitcatg 27 OO ttctgg tagt agtttcCagg aggtoaacgc gctt catagt tacgttgacc atgttgatga 276 O taaagagt cc ggctataatt c tagggctta t cott catac acggacacca tagaaggttt 282O aaaggt catg gaattgat C 2839
<210s, SEQ ID NO 5 &211s LENGTH: 1773 &212s. TYPE: DNA <213> ORGANISM: Malus x domestica
<4 OOs, SEQUENCE: 5 aacct tacac ttcttctaca attctaagtt to catttitcc aac atcc cat cacattgttc 6 O aaaaatat catcagoctoga gttagggittt attatcc titc gtgaccctic c titt tag tatt 12 O tggttcttitt togaaagacga tttgttaggt gtttctaggc citctago cat cittgtat citc 18O accaaaaaaa aatatattac cagctgttta caccalaatta aatagtaaaa agaaagcatc 24 O aatggctitta aaaacacagt togttgttggtc atttgttgtt gttitttgttg titt cott cag 3OO tacaact tca tottctggta gtag titt.cca ggaggtoaac gcgctt cata gttacgttga 360 c catgttgat gataaagagt ccggctataa ttctagggct tat cott cat acacggacac 42O Catagalaggt ttalaaggt ca taattgat Caggccalaga act cagctct t cagttcaag 48O gaagct Caac acaat Caccg gtgggatago alacat catca gct Coggcca aaac Cattag 54 O cgt.cgacgat tittggagcta aagggaatgg totgatgac acacaggcat ttgttgaaggc 6OO atggaaggca gcttgttctt C cagtggagc tatggttctt gtggtaccac agaagaact a 660
US 2011/O 191902 A1 Aug. 4, 2011 27
- Continued gtgcaataac ttggtggtga agaatctgaa tat Cogagac goacaacaaa to atgtc. 958
<210s, SEQ ID NO 7 &211s LENGTH: 653 &212s. TYPE: DNA <213> ORGANISM: Malus Sieboldii
<4 OO > SEQUENCE: 7 t catgtgtca aatacccaga atat cactat citccagotcc gttataggaa Caggtgatga 6 O ctgt atttct attgtgagtg ggit cocaaag agttcaa.gcc acaga catta Cttgttggacc 12 O aggc catgga at Cagt attg gtagcttggg agaagacggc ticaaaagat C atgttt Cagg 18O agtatttgttgaatggagcta agctitt Cagg aacct coaat ggact Cogga t calagacgtg 24 O ggagggaggc ticaggcagtg caaccaac at tittitt C cag aatgtgcaaa tgaacgatgt 3OO cgc.caa.cccc atcatcatcg accagaacta ccgtgaccac aaaac caaag attgcaaaca 360 acagaaatcg gcggttcaag taaaaatgt gttgtacaaa alacatalaga.g gaacgagtgc 42O titc.ccg.cgac gcgataacgt taactgcag cccaagttgtt CCttgtcagg ggat.cgtgct 48O gcaaagtgtt Caactgcaga atggalaga.gc tigaatgcaac aatgtta agc ctgcttacaa 54 O aggagttgtc. tcc cct agat gttaaaacct agggct cata attatgggca atgtgaaaca 6OO gct tatgcaa ttcttgtacg aattagcaca taaataattg tttgtttgta ata 653
1-60. (canceled) b) the sequence of SEQID NO: 4, 61. A method for producing a plant with fruit having c) a sequence with at least 70% identity to the sequence of increased post-harvest storage life, the method comprising SEQID NO:5, and reducing the expression or activity in the plant, of a polypep d) the sequence of SEQID NO: 5. tide with the amino acid sequence of SEQ ID NO: 1, or a 66. The method of claim 64, wherein the polynucleotide variant of the polypeptide with at least 70% identity to the comprises at least 15 contiguous nucleotides that are at least amino acid sequence of SEQID NO: 1, wherein the method 70% identical to part of the endogenous gene or nucleic acid. comprises the step of introducing a polynucleotide into a 67. The method of claim 64, wherein the polynucleotide plant cell, or plant, to effect reducing the expression of the comprises at least 15 contiguous nucleotides that hybridise polypeptide or variant. under stringent conditions to the endogenous gene or nucleic 62. The method of claim 61, wherein the fruit have at least acid. one of: 68. The method of claim 64, wherein the polynucleotide is a) increased firmness, introduced into the plant as part of a genetic construct. b) reduced water loss, 69. The method of claim 67, wherein the genetic construct c) reduced cell separation, is an expression construct comprising a promoter operably d) increased juiciness, linked to the polynucleotide. e) increased crispiness, 70. The method of claim 68, wherein the polynucleotide in f) increased waxiness, and an antisense orientation relative to the promoter. g) reduced Susceptibility to necrophytic pathogens, during, 71. The method of claim 61, wherein the polynucleotide or after, post-harvest storage. comprises at least one of: 63. The method of claim 61, wherein the variant has a) at least 15 contiguous nucleotides of a sequence with at polygalacturonase activity. least 70% identity to the sequence of SEQID NO: 4, 64. The method of claim 61, wherein the polynucleotide b) at least 15 contiguous nucleotides of the sequence of comprises at least one of: SEQID NO:4, i) a sequence with at least 70% identity to part of an endog c) at least 15 contiguous nucleotides of a sequence with at enous gene, or nucleic acid, that encodes the polypeptide least 70% identity to the sequence of SEQID NO: 5, or variant thereof, and d) at least 15 contiguous nucleotides of the sequence of ii) a sequence that hybridises under stringent conditions to SEQ ID NO:5. part of an endogenous gene, or nucleic acid, encoding 72. The method of claim 61, wherein a plant with reduced the polypeptide, or a variant of the polypeptide. expression of the polypeptide is regenerated from the plant 65. The method of claim 64, wherein the endogenous gene cell. comprises at least one of: 73. A plant produced by the method of claim 61, wherein a) a sequence with at least 70% identity to the sequence of the plant is genetically modified to contain the polynucle SEQID NO:4, otide. US 2011/O 191902 A1 Aug. 4, 2011 28
74. An expression construct comprising a promoter oper 81. The plant of claim 79, wherein the fruit have increased ably linked to a polynucleotide comprising at least one of firmness during, or after, post-harvest storage. i) a fragment of at least 15 contiguous nucleotides of a 82. An isolated polynucleotide encoding a polypeptide sequence with at least 70% identity to any one of SEQID comprising a sequence of SEQ ID NO: 2 or 3 or a variant NO: 4, 5, 6 and 7, wherein the sequence with 70% thereof, wherein the variant has polygalacturonase activity identity to any one of SEQID NO: 4, 5, 6 and 7, encodes and comprises a sequence with at least 96% identity to SEQ a polypeptide with polygalacturonase activity, and ID NO: 2 or 3. wherein the polynucleotide is in an antisense orientation 83. The isolated polynucleotide of claim 82 comprising the relative to the promoter, and sequence of SEQID NO: 6 or 7, or a variant thereof, wherein ii) a fragment of at least 15 contiguous nucleotides any one the variant encodes a polypeptide with polygalacturonase of SEQID NO: 4, 5, 6 and 7. activity, and comprises a sequence with at least 96% identity 75. The expression construct of claim 74 which is an RNAi to SEQID NO: 6 or 7. COnStruct. 84. An isolated polypeptide comprising the amino acid 76. A plant cell, or plant, comprising an expression con sequence of SEQID NO: 2 or 3, or a variant thereof, wherein struct of claim 14. the variant has polygalacturonase activity, and comprises a 77. The plant cell, or plant, of claim 76 that has modified sequence with at least 96% identity to SEQID NO: 2 or 3. expression of the endogenous nucleic acid corresponding to 85. A genetic construct, expression construct, or RNAi the polynucleotide. construct, which comprises a polynucleotide of claim 82. 78. The plant cell, or plant, of claim 77, wherein the endog enous nucleic acid encodes a polypeptide with polygalactur 86. The construct of claim 85 which comprises a promoter onase activity. linked to the polynucleotide. 79. The plant of claim 76 that has, or is capable of produc 87. A host cell, plant cell, or plant genetically modified to ing, fruit with increased post-harvest storage life. express a polynucleotide of claim 82. 80. The plant of claim 79, wherein the fruit have at least one 88. A host cell, plant cell, or plant comprising the construct of: of claim 85. a) increased firmness, 89. A plant part, seed, fruit, propagule or progeny of a plant b) reduced water loss, of claim 87 that is genetically modified to contain the poly c) reduced cell separation, nucleotide. d) increased juiciness, 90. A plant part, seed, fruit, propagule or progeny of a plant e) increased crispiness, of claim 88 that is genetically modified to contain the f) increased waxiness, and COnStruct. g) reduced Susceptibility to necrophytic pathogens, during, or after, post-harvest storage.