AU778691B2 - Genes coding for tomato beta-galactosidase polypeptides - Google Patents

Description

WO 99/64564 PCT/US99/12697 GENES CODING FOR TOMATO P-GALACTOSIDASE

POLYPEPTIDES

Field of the Invention The present invention relates to a family of novel plant genes encoding polypeptides characterized by their ability to hydrolyze terminal non-reducing P-D-galactosyl residues from P-D-galactosides. More specifically, a polynucleotide sequence derived from a cDNA clone designated pZBG2-1-4 (referred to in U.S. Provisional Appln. No. 60/088,805 as pTompgal which encodes a specific plant polypeptide named p-galactosidase II, is provided.

Also provided are cDNA clones encoding six other homologous polypeptides, methods of using these cDNA clones for producing P-D-galactoside polypeptides of the invention, and methods of modifying fruit quality by employment of a polynucleotide or polypeptide of the present invention.

Background of the Invention The most conspicuous and important processes related to post-harvest quality of climacteric fruit are the changes in texture, color, taste, and aroma which occur during ripening. Because of the critical relationship that deleterious changes in texture have to quality and post-harvest shelf-life, emphasis has been placed on studying the mechanisms involved in the loss of firmness that occurs-during tomato fruit ripening. Although fruit softening may involve changes in turgor pressure, anatomical characteristics and cell WO 99/64564 PCT/US99/12697 2 wall integrity, it is generally assumed that cell wall disassembly leading to a loss of wall integrity is a critical feature. The most apparent changes, in terms of composition and size, occur in the pectic fraction of the cell wall (see references in Seymour and Gross, 1996).

Changes known to occur in the pectic fraction of the cell wall during fruit ripening include increased solubility, depolymerization, de-esterification and a significant net loss of neutral sugar containing side chains (Huber, 1983; Fischer and Bennett, 1991; Seymour and Gross, 1996). The best characterized pectin-modifying enzymes are polygalacturonase (endo-al-4-D-galacturonan hydrolase; E.C. 3.2.1.15; PG) and pectin methylesterase 3.1.1.11; PME).

Although PG and PME are relatively abundant and have substantial activity during tomato fruit ripening, softening still occurs, albeit with a slight delay, in fruit where PG (Smith et al. 1988, 1990) or PME (Tieman et al. 1992; Hall et al. 1993) gene expression and enzyme activity was significantly downregulated in transgenic plants. Moreover, over-expression of PG in nonripening mutant rin tomato fruit did not result in softening even though depolymerization and solubilization of pectin was evident (Giovannoni et al., 1989).

Among the other known pectin modifications that occur during fruit development, one of the best characterized is the significant net loss of galactosyl residues which occurs in the cell walls of many ripening fruit (Gross and Sams, 1984; Seymour and Gross, 1996). Although some loss of galactosyl residues could result indirectly from the action of PG, p-galactosidase (exo- Rl( 4) .galactopyranoside; E.C. 1,2.123) is the only enzyme identified in J rvl g 'u

J

WO 99/64564 PCT/US99/12697 higher plants capable of directly cleaving p(l-4)galactan bonds, and probably plays a role in galactan sidechain loss (DeVeau et al., 1993; Carey et al., 1995; Carrington and Pressey, 1996). No endo-acting galactanase has yet been identified in higher plants. The view that p-galactosidase is active in releasing galactosyl residues from the cell wall during ripening is supported by the dramatic increase in free galactose, a product of p-galactosidase activity (Gross, 1984) and a concomitant increase in activity of a particular enzyme, designated P-galactosidase II, in tomatoes during ripening (Carey et al., 1995).

P-galactosidase activity is thought to be important in cell wall metabolism (Carey et al., 1995). P-Galactosidases are generally assayed using artificial substrates such as p-nitrophenyl-p-D-galactopyranoside (PNP), 4- Smethylumbelliferyl-p-D-galactopyranoside and 5-bromo-4-chloro-3-indoxyl- P-D-galactopyranoside (X-GAL). However, it is clear that p-galactosidase

DI

is also active against natural substrates, P (l-4)galactan (Carey et al., 1995; Carrington and Pressey, 1996; Pressey, 1983). P-Galactosidase proteins have been purified and characterized in a number of other fruits including kiwifruits (Ross et al., 1993), coffee (Golden et al., 1993), persimmon (Kang et al., 1994), and apple (Ross et al., 1994).

Carey et al. (1995) were able to purify three previously identified 3galactosidases from ripening tomato fruit (Pressey, 1983), but only one (Pgalactosidase II) was active against p(l--4)galactan. Even though they were able to identify putative p-galactosidase cDNA clones, none of the cDNA's deduced amino acid sequences matched the amino terminal sequence of the 3galactosidase II protein. Although 3-galactosidase II, a protein present in WO 99/64564 PCT/US99/12697 tomato (Lycopersicon esculentum Mill.) fruit during ripening and capable of degrading tomato fruit galactan has been purified, cloning of the corresponding gene has been elusive.

The modification of plant gene expression has been achieved by several methods. The molecular biologist can choose from a range of known methods to decrease or increase gene expression or to alter the spatial or temporal expression of a particular gene. For example, the expression of either specific antisense RNA or partial (truncated) sense RNA has been utilized to reduce the expression of various target genes in plants (as reviewed by Bird and Ray, 1991, Biotechnology and Genetic-Engineering Reviews 9:207-227). These techniques involve the incorporation into the genome of the plant of a synthetic gene designed to express either antisense or sense RNA. They have been successfully used to down-regulate the expression of a range of individual genes involved in the development and ripening of tomato fruit (Gray et al, 1992, Plant Molecular Biology, i9:69-87). Methods to increase the expression of a target gene have also been developed. For example, additional genes designed to express RNA containing the complete coding region of the target gene may be incorporated into the genome of the plant to "over-express" the gene product. Various other methods to modify gene expression are known; for example, the use of alternative regulatory sequences. The complete disclosure of each of the references cited above is fully incorporated herein by reference.

The need therefore exists to clone a gene for 3-galactosidase II and related polype-pties, and using known methods of modification of plant gene expression, thereby to provide methods for modifying quality of fruits, particularly by modifying the cell wall, thereby directly affecting the ripening of the fruit.

In the specification and claims the term "comprising" shall be understood to have a broad meaning similar to the term "including" and will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. This definition also applies to variations on the term "comprising" such as "comprise" and "comprises".

Summary of the Invention The present invention is based on the discovery of novel DNA sequences derived from cDNA clones from a family of genes encoding p-galactosidases.

The phylogenic tree based on the shared amino acid sequence identities for the DNA sequences of the present invention is shown in Figure 1A, B. Five cDNA and two RT-PCR clones, designated herein as TBG 1, TBG2, TBG3, TBG4, 15 TBG5, TBG6, and TBG7 and having the nucleic acid sequences designated SEQ ID NOs 1-7, respectively as shown in Figure 2, were identified which had a high degree of shared sequence identity to other known p-galactosidases. The corresponding amino acid sequences are designated herein as SEQ ID NOs 8- 14, respectively and are shown in SEQ ID NOs 1-7 are recorded Accessions Numbers: SEQ ID NO: 1 TGB1 SEQ ID NO: 2 TGB2 SEQ ID NO: 3 TGB3 SEQID NO: 4 TGB4 SEQ ID NO:5 TGB5 SEQID NO: 6 TGB6 SEQ ID NO: 7 TGB7 Figure 2 and 3. The nucleotide sequences for in Gen Bank with the following respective AF023847 AF154420 AF154421 AF020390 AF154423 AF154424 AF154422 deposit Sept 10,1997 deposited May 19,1999 deposited May 20,1999 deposited Aug 21,1997 deposited May 20,1999 deposited May20,1999 deposited May 20,1999 WO 99/64564 PCT/US99/12697 O 6 Throughout the following discussion, wherever TBG4 is indicated in the description of the invention, it is to be understood that TBG1-3 and 5-7 are also to be included in that description, unless otherwise indicated.

A method of providing a DNA sequence of the invention, either by cloning a cDNA (for instance, pZBG2-1-4) that codes for a protein of the present invention, such as 3-galactosidase II, or by deriving the DNA sequence from genomic DNA, or by synthesis of a DNA sequence ab initio using the cDNA sequence as a guide is also provided.

A method for modifying cell wall metabolism which involves modifying the activity of at least one galactosidase, and thus modifying the quality of the fruit is also provided.

Also provided by the present invention is a DNA construct including some or all of an exemplary 3-galactosidase DNA sequence under control of a transcriptional initiation region operative in plants, so that the construct can generate RNA in plant cells.

Also discovered is an enhancer/promoter associated with expression of the genes encoding 3-galactosidase.

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of P-galactosidase polypeptides or peptides by recombinant techniques.

WO 99/64564 PCTIUS99/12697 The present invention also provides plant cells containing DNA constructs of the present invention; plants derived therefrom having modified P-galactosidase gene expression; and seeds produced from such plants.

The P-galactosidase II protein of the present invention has demonstrated enzyme activity in cell wall disassembly leading to loss of tissue integrity and fruit softening. The P-galactosidase II protein also may be involved in cell wall turnover, which could be involved in cell extension and/or expansion and therefore plant growth and development.

By hydrolyzing galactose from the cell wall, the enzyme may allow ripening to commence and/or progress, since galactose may be involved in stimulating ethylene production alone or in conjunction with unconjugated Nglycans.

The P-galactosidase of the invention may be involved in conversion of chloroplasts (green chlorophyll) to chromoplasts (red lycopene) during fruit ripening by degrading chloroplast membrane galactolipids.

The family of genes represented by the nucleotide sequences shown in Figure 2 is expected to code for a group of similar enzymes with the same type of hydrolytic activity but with different tissue and/or substrate specificity's or cellular compartmentation profiles.

The -galactosidase I protein of the present invention as well as other proteins encoded in the nucleotide sequences shown in Figure 2 may be used for preparation of pectin and other cell wall derived polymers with lowered galactosyl content for use in biofilms and solutions (for example in 8 clarification of fruit juices) requiring lower or higher cross-linking or viscomertric properties.

The present invention also provides P-galactosidase enzymes for use as components of enzyme mixtures for protoplast isolation.

Brief Description of the Figures Figure 1A and 1B shows a phylogenic tree based on shared amino acid sequence identity among tomato P-galactosidase clones TGB1-7 and other known plant P-galactosidase polypeptides.

Figure 2 shows cDNA sequences [SEQ ID NOs: 1-7, respectively] for the seven P-galactosidase genes of the invention: TGB1, TGB2, TGB3, TGB6, TGB7.

o9 99* Figure 3 shows multiple sequence alignment of the deduced amino acid sequences of tomato fruit for cDNA clones TGB1, TGB2, TGB3, TGB4, TGB6 and TGB7 [SEQ ID NOs: 8-14, respectively] and various plant Pgalactosidase cDNA clones.

Figure 4 shows autoradiograph of northern blot analysis of TBG expression in various plant tissues (flowers, leaves, roots and stems).

99 Figure 5 shows Autoradiograph of northern blot analysis of TBG expression in fruit tissues at different stages of development.

WO 99/64564 PCT/US99/12697 9 Figure 6 shows autogradiograph of northern blot analysis of TBG expression in fruit tissues (mature green or turning stage fruit peel, outer pericarp, inner paricarp and locular).

Figure 7 shows autoradiograph of northern blot analysis of TBG expression in normal and mutant fruit tissues.

Figure 8 shows autoradiograph of northern blot analysis of TBG expression in response to ethylene treatment of mature green fruit tissues.

Figure 9 shows Western blot analysis of TBG4 expression by yeast.

Figure 10 shows detection of P-galactosidase activity from pZBG2-1-4 expression in E. coli.

Figure 11 A E shows the comparative results of texture measurements for fruit from tomato plants containing antisense constructs to suppress TBG4 mRNA and fruit from the parental line.

Figures 12A B show Northern blot analysis of TBG4 expression in transgenic fruit containing TBG4 antisense construct.

Figure 13 shows a Binary construct used to transform plants and express TBG4 (pZBG2-1-4) in the antisense orientation.

WO 99/64564 PCT/U S99/1 2697 Detailed Description The following detailed description is directed to a preferred embodiment of the present invention and is intended as illustrative of each of other DNA sequences of the present invention.

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding P-galactosidase polypeptides, particularly a p-galactosidase II polypeptide having the amino acid sequence shown in Figure 2. The DNA sequence of the exemplary p-galactosidase II cDNA clone of the invention, which was determined from a cDNA clone, pZBG2-1-4, encoding P-galactosidase II, is recorded in GenBank as Accession Number AF020390. Not all p-galactosidases possess in vitro activity against extracted cell wall material via the release of galactose from wall polymers containing p(l-4)-D-galactan. The polypeptide expressed from the exemplary p-galactosidase II clone, pZBG2-1-4, has been shown to exhibit p-galactosidase activity and exogalactinase activity.

The exemplary P-galactosidase II protein of the present invention, as shown in Figure 2, shares sequence homology with the amino acid sequence deduced from p-galactosidase cDNA clones of TBG2-7 and cDNA clones of the fruits of asparagus (accession number P45582), apple (accession number P48981), and carnation (accession number Q00662), as well as with Pgalactosidase cDNA clones of a previously published sequence of a tomato Igalactosidase cDNA clone designated pTompgall (accession number P48980) isolated from ripe 'Ailsa Craig' fruit (Carey et al., 1995). The ORF of the clone TBG1 disclosed herein by the inventors (accession number AF023847) WO 99/64564 PCT/US99/12697 11 is nearly identical to the cDNA previously described by Carey et al. As shown in Figure 2, the shared deduced sequence identity is high among all the published plant p-galactosidases of the seven clones (TBG1-7) and the other plant p-galactosidases.

BLAST searches of the database also indicated significant shared sequence identity between domains of the plant p-galactosidases and mammalian and fungal p-galactosidases, however little share sequence identity was detected with bacterial p-galactosidases.

As shown in Figure 1, the shared amino acid identity of TBG 1 and TBG3 was high. TBG4 was also very similar to both TBGI and 3. The amino acid sequences of TBG2 and 7 were unique because several regions of amino acid insertions appear throughout their sequence (Figure 3).

Nucleic Acid Molecules Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using a PCR-based dideoxynucleotide terminator protocol and an ABI automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc., Foster City, CA), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least WO 99/64564 PCT/US99/12697 12 about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.

By "nucleotide sequence" of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides G, C and where each thymidine deoxyribonucleotide in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine Using the information provided herein, such as the exemplary nucleotide sequence shown in Figure 2 [SEQ ID NO: a nucleic acid molecule of the present invention encoding a P-galactosidase II polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in Figure 2 [SEQ ID NO: 4] was discovered in a cDNA library derived from breaker, turning and pink fruit pericarp from 'Rut er' tomat plants.

t_ lnnt.- WO 99/64564 PCT/US99/12697 13 The complete sequence of the cDNA insert of pZBG2-1-4 is accessible in the GenBank (no. AF020390) and is provided in Figure 2 [SEQ ID NO: 4].

The cDNA insert is 2532 nucleotides (nt) long and contains a single, long open reading frame (ORF) predicted to start with the first in-frame ATG at nt 64 and end with TAA at nt 2238. This ORF codes for a 79 kD protein 724 amino acids long. The deduced amino acid sequence of pZBG2-1-4 shared significant amino acid identity to all published plant p-galactosidase sequences in the database (Figure 1A,B). When the entire ORF of each p-galactosidase gene was compared to pZBG2-1-4, the shared sequence identity was about 64% for tomato pTompgal 1 (P48980), about 67.6% for apple (P48981), about 63% for asparagus (P45582) and about 55% for carnation (Q00662). As one of ordinary skill would appreciate, due to the possibilities of sequencing errors discussed above, the actual complete p-galactosidase II polypeptide encoded by the deposited cDNA, which comprises about 724 amino acids, may be somewhat longer or shorter. More generally, the actual open reading frame may be anywhere in the range of ±20 amino acids, more likely in the range of amino acids, of that predicted from either the first methionine codon from the N-terminus shown in Figure 2 [SEQ ID NO: In any event, as discussed further below, the invention further provides polypeptides having various residues deleted from the N-terminus of the complete polypeptide, including polypeptides lacking one or more amino acids from the N-terminus of the Pgalactosidase II polypeptide described herein.

14 Leader and Mature Sequences Analysis of the deduced amino acid sequence of pZBG2-1-4 suggested a high probability for secretion based on the presence of a hydrophobic leader sequence, a leader sequence cleavage site and three possible N-glycosylation sites. The programs PSORT V6.4 (Nakai and Kanehisa, 1992, incorporated herein by reference) and SignalP V 1.1 (Nielsen et al., 1997, incorporated herein by reference), were used to predict that the ORF contains a hydrophobic leader sequence that would be cleaved between the alanine and serine residues at positions 23 and 24 respectively, and that the mature polypeptide has an extracellular location. The mature polypeptide contains three possible Nglycosylation sites at asparagine numbers 282, 459 and 713, however the asparagine at position 713 is unlikely to be glycosylated due to the proline at position 714. The predicted molecular mass of the unglycosylated mature polypeptide was 75 kD with a pi of 8.9.

Accordingly, the amino acid sequence of the complete p-galactosidase II protein of the invention includes a leader sequence and a mature protein, as shown in Figure 3 [SEQ ID NO: 11]. More in particular, the present invention provides nucleic acid molecules encoding a mature form of the p-galactosidase II protein. Thus, according to the signal hypothesis, secreted proteins have a signal or secretory leader sequence which is cleaved from the complete polypeptide to produce a secreted "mature" form of the protein. In some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that the cleavage specificity of a secreted protein is ultimately determined by the WO 99/64564 PCT/US99/12697 primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide. Therefore, the present invention provides a nucleotide sequence encoding the mature p-galactosidase II polypeptide having the amino acid sequence encoded by the cDNA shown in Figure 2 [SEQ ID NO: 4] and provided in GenBank (Accession No. AF20390). By the "mature p-galactosidase II polypeptide having the amino acid sequence encoded by the cDNA clone shown in Figure 2 [SEQ ID NO: 4] is meant the mature form(s) of the p-galactosidase II protein produced by expression in a plant cell of the complete open reading frame encoded by the cDNA sequence of the clone shown in Figure 2 [SEQ ID NO: 4] and provided in GenBank (Accession No. AF20390).

The exemplary P-galactosidase II cDNA of the present invention (TBG4) has been expressed in E. coli strain XLI blue MR (lacZ) (Stratagene, La Jolla, CA), as described hereinbelow (see Example).

Analysis of the deduced amino acid sequence of cDNA clones representing the other p-galactosidase genes of the invention also revealed open reading frames and, in some cases, suggested a high probability for secretion of the encoded proteins. All the full-length cDNA clones were predicted to have a signal sequence (Fig. Using the two prediction programs SignalP and PSORT, TBG4 was predicted to be secreted by both programs. TBG1, 2 and 3 were predicted to have cleavable signal sequences by SignalP, but uncleavable signal sequences by PSORT. TBG7 was suggested to be targeted to the chloroplast by PSORT. Particular observations for each of the seven clones are as follows, based on the presence of a hydrophobic WO 99/64564 PCT/US99/12697 leader predicted by the programs PSORT V6. and SignalP V 1.1: TBG 1: initiation codon at 306 [SEQ ID NO: ORF 835 amino acids [SEQ ID NO: signal sequence at 1-24; TBG2: initiation codon not determined [SEQ ID NO: ORF 888 amino acids [SEQ ID NO: signal sequence at 1-25; TBG3: initiation codon at 32 [SEQ ID NO: ORF 838 amino acids [SEQ ID NO: 10], signal sequence at 1-22; TBG5: initiation codon not determined [SEQ ID NO:5], ORF 251 amino acids [SEQ ID NO: 12], signal sequence not determined; TBG6: initiation codon not determined [SEQ ID NO:6], ORF 248 amino acids [SEQ ID NO: 13], signal sequence not determined; TBG7: initiation codon at 104 [SEQ ID NO: ORF 870 amino acids [SEQ ID NO:14], signal sequence at 1-35.

The deduced amino acid sequences of the seven clones was also subjected to analysis using the program DNAsis and the predictions for molecular mass, cellular targeting, pi and potential N-linked glycosylation sites are summarized in Table I.

WO 99/64564 PCT/US99/12697 Table I. Tomato P-galactosidase (TBG) cDNA sequence data. Five full-length and 2 partial-length cDNAs were cloned and sequenced.

The DNA and deduced amino acid sequence data is presented below CLONE mRNA(kb) kD pl N-LINK TARGET TBG1 3.2 90.8 6.2 2 ER/OUT TBG2 3.0 97.0 6.2 6 PM TBG3 2.8 90.5 8.2 1 ER/OUT TBG4 2.6 77.9 8.9 3 OUT -3 TBG6 -3 TBG7 3.0 93.3 8.0 6 CHLOR N-LINK possible N-linked glycosylation sites; ER endoplasmic reticulum; out secreted; PM tethered to plasma membrane; CHLOR chloroplast As indicated, nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded.

Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.

By "isolated" nucleic acid molecule(s) is intended a nucleic acid .oleule, DT A or RNNA, which has been removed from its native environment IIlV I lACUI. A .Pl _t WO 99/64564 PCT/US99/12697 18 For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) with an initiation codon at position 64 of the nucleotide sequence shown in Figure 2 [SEQ ID NO: 4].

Also included are DNA molecules comprising the coding sequence for the mature 3-galactosidase II protein shown at positions 135-2532 of Figure 2 [SEQ ID NO: 4].

In addition, isolated nucleic acid molecules of the invention include DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the P-galactosidase I protein. Of course, the genetic code and speciesspecific codon preferences are well known in the art. Thus, it would be routine for one skilled in the art to generate the degenerate variants described above, for instance, to optimize codon expression for a particular host change codons in the plant mRNA to those preferred by a bacterial host such as E. coli). Preferably, this nucleic acid molecule will encode the mature polypeptide encoded by the above-described deposited cDNA clone.

WO 99/64564 PCT/US99/12697 19 The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figure 2 [SEQ ID NO: 4] or a nucleic acid molecule having a sequence complementary to the above sequence. Such isolated molecules, particularly DNA molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the p-galactosidase II gene in plant tissue, for instance, by Northern blot analysis.

The present invention is further directed to nucleic acid molecules encoding portions of the nucleotide sequences described herein as well as to fragments of the isolated nucleic acid molecules described herein. In particular, the invention provides a polynucleotide having a nucleotide sequence representing the portion of Figure 2 [SEQ ID NO: 4] which consists of positions 1-2538 of Figure 2 [SEQ ID NO: 4].

In addition, the invention provides additional nucleic acid molecules having nucleotide sequences related to extensive portions of Figure 2 [SEQ ID NO: 4] which have been determined from the following related cDNA clones: TBG1-3 and TBG5-7 as shown in Figure 3, SEQ. NO's 1-3 and 5-7 In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the cDNA clone shown in Figure 2 [SEQ ID NO: By "stringent hybridization conditions" is intended overnight incubation at 42° C in a solution comprising: formamide. 5x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 5x Denhardt's solution, 10% dextran sulfate, and 20 pg/ml WO 99/64564 PCT/US99/12697 denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1x SSC at about 65° C.

As indicated, nucleic acid molecules of the present invention which encode a P-galactosidase II polypeptide may include, but are not limited to those encoding the amino acid sequence of the mature polypeptide, by itself; and the coding sequence for the mature polypeptide and additional sequences, such as those encoding the about 1-23 amino acid leader sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences.

Also discovered is an enhancer/promoter associated with expression of the genes encoding p-galactosidase. The inventors have characterized the expression profile of TBG2 mRNA and have cloned a lambda genomic cDNA.

TBG2 is expressed before the onset of fruit ripening and continues at uniform level throught all the ripening stages. TBG2 has been found to be expressed in all fruit tissues and has also been found to be fruit specific. Experiments have shown TBG2 to be unaffected by ethylene. TBG2 is expressed in the ripening mutants rin, nor and Nr at the normal chronological time after anthesis. The promoter discovered would be useful to express any gene in the sense or antisense orientation, specifically in tomato fruit, in all tomato fruit tissues, starting before and continuing throughout the entire ripening process. The promoter could also be used to express any gene in the ripening mutants rin, nor and Nr without the need to gas the fruit with exogenous ethylene.

WO 99/64564 PCT/US99/1269 7 21 Variant and Mutant Polynucleotides The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the p-galactosidase II protein. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, ed., John Wiley Sons, New York (1985).

Non-naturally occurring variants may be produced using art-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the Pgalactosidase II protein or portions thereof. Also especially preferred in this regard are conservative substitutions.

Most highly preferred are nucleic acid molecules encoding the mature protein having the amino acid sequence shown in Figure 2 as pZBG2-1-4 or the mature p-galactosidase II amino acid sequence encoded by the deposited cDNA clone.

Further embodiments include an isolated nucleic acid molecule comprising a polynucieotide having a nucleotide sequcnce at least WO 99/64564 PCT/US99/12697 22 identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to a polynucleotide selected from the group consisting of: a nucleotide sequence encoding the p-galactosidase II polypeptide having the complete amino acid sequence in Figure 2 [SEQ ID NO: 4] a nucleotide sequence encoding the mature p-galactosidase II polypeptide shown in Figure 2 [SEQ ID NO: a nucleotide sequence complementary to any of the nucleotide sequences in or above.

Vectors and Host Cells The present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of Pgalactosidase II polypeptides or fragments thereof by recombinant techniques.

The vector may be, for example, a phage, plasmid, viral or retroviral vector.

Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of WO 99/64564 PCT/US99/12697 23 retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.

Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, StrepZBG2-1-4yces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293 and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, and pQE-9, available from QIAGEN, Inc., supra; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNHI8A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

PCT/US99/12697 WO 9Q/64564 24 Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986).

Example Tomato (Lycopersicon esculentum Mill., cv. 'Rutgers') plants were grown in a greenhouse using standard cultural practices. The ripening mutants, ripening inhibitor (rin), non-ripening (nor) and never ripe (Nr) (Tigchelaar et al., 1978), were all in the 'Rutgers' background. Flowers were tagged at anthesis and fruit were harvested according to the number of days postanthesis (dpa) or based on their surface color using ripeness stages as previously described (Mitcham et al., 1989), the complete disclosure of which is hereby fully incorporated herein by reference. For gene expression studies, a variety of leaf, flower, and stem tissues were harvested from greenhousegrown plants and roots were harvested from seedlings grown in basal tissue culture medium for 4 weeks after seed germination.

RNA Extraction Fruits were processed immediately after harvest in the greenhouse by chilling on ice, excising the various tissues and freezing them in liquid nitrogen. Tissue samples were ground using a mortar and pestle and stored at 0 C. RNA was extracted using the method described in Verwoerd et al.

(1989). Poly(A)RNA was purified from total RNA using oligo(dT) columns WO 99/64564 PCT/US99/12697 (Pharmacia, Piscataway, NJ). RNA was quantified by measuring A 260 using a dual beam spectrophotometer.

RT-PCR

Degenerate primers were designed based on the highest shared deduced amino acid sequence identity we found between an apple (accession number P48980), asparagus (P45582) and carnation (Q00662) P-galactosidase cDNA clones. The two primers used for the first reaction were BG5'E1 (WSNGGNWSNATHCAYTAYCC) and BG3'E (CCRTAYTCRTCNADNGGNGG). A second reaction was done on the products of the first reaction using (ATHCARACNTAYGTNTTYTGG) and BG3'E. The degeneracy code for the primer sequences is N=a+t+c+g; H=a+t+c; B=t+c+g; D=a+t+g; V=a+c+g; R=a+g; Y=c+t; M=a+c; K=t+g; S=c+g; and W=a+t. The 5' and 3' primers corresponded to amino acids 72-78 and 321-315 of the apple clone, respectively. Amplification was done using AmpliTaq DNA polymerase (Perkin Elmer, Norwalk, CT) and standard PCR conditions using the cDNA made for the first cDNA library described below as a template (Ausubel et al., 1987). PCR products were separated in an agarose gel and fragments of the expected size (approximately 750 bp) were purified, cloned into pCRscript (Stratagene, La Jolla, CA) and sequenced.

cDNA library Two cDNA libraries were constructed. The first comprised poly(A) RNA isolated from breaker, turning and pink fruit pericarp from 'Rutgers' plants.

WO 99/64564 PCT/US99/12697 26 The cDNA synthesis and library construction was done exactly according to the manufacturers instructions for the ZAP-cDNA Gigapack II Gold Cloning Kit (Stratagene), the complete disclosure of which is fully incorporated herein by reference. First-strand cDNA synthesis was primed using a poly(dT) primer and inserts were directionally cloned into the Uni-Zap XR vector using EcoRI and Xhol restriction sites. The second library comprised poly(A) RNA isolated from all fruit tissues (except seeds) from immature green, mature green, breaker, turning, pink, red-ripe and over-ripe fruit of 'Rutgers' plants.

The cDNA synthesis and library construction was done exactly according to the manufacturers instructions for the SuperScript Lambda System for cDNA synthesis and Cloning (GibcoBRL, Gaithersburg, MD). First-strand cDNA synthesis was primed using a oligo(dT) primer and cDNA inserts were directionally cloned into the ZipLox cloning vector using Sal and Notl restriction sites. Both libraries were amplified and maintained using the host strains provided by the manufacturer, according to their instructions.

One of the clones (RT-PCR2-1) was used to screen 10 6 plaques from the tomato fruit cDNA libraries at low stringency (hybridization at 45 0 C, no formamide and final wash with 0.2X SSC at 42 0 Thirty positive cDNA clones were identified and partially sequenced. Complete sequencing and characterization of the RT-PCR and cDNA clones revealed the possibility of seven unique p-galactosidase genes.

WO 99/64564 PCT/US99/12697 27 DNA and RNA Gel Blot Analysis Southern analysis was done using the 3' UTR of each full length clone and the RT-PCR clones as probes against restriction enzyme digested genomic DNA. DNA gel blot analysis was done essentially as described in Smith and Fedoroff (1995) except that 3 gg of genomic DNA was used for each digest.

The genes corresponding to the clones appeared to be present as single copies (data not shown). The same probes were used to map 6 of the 7 genes using RFLPs of recombinant inbred lines and the loci names and map positions are shown in Table II (James Gioviannone, Texas A&M University, personal communication).

Table II. TBG loci map positions. Genes were maped by Southern analysis using RFLPs of recombinant inbred lines.

Gene chromosome map position TBG1 12* overlap of IL 12-2, IL 12-3 TBG2 9 IL 9-3 TBG3 3 IL TBG4 12* overlap of IL 12-2, IL 12-3 11 IL 11-3 TBG6 2 overlap of IL 2-4, IL TBG7 no RFLP *TBG1 and 4 are loosely linked Total RNA (20 pgg/ lane) was separated in a formaldehyde/Mops agarose gel, transferred to Hybond-N' nylon membrane (Amersham, Arlington Heights, IL), fixed by incubating for 2 h at o80 1hbridized overnight in a WO 99/64564 PCT/US99/12697 28 hybridization incubator (Robbins Scientific, Sunnyvale, CA) using a buffer described by Church and Gilbert (1984) washed to a final stringency of 0.1 X SSC with 0.2% SDS at 65 0 C, and autoradiographed essentially as described by Ausubel et al. (1987). An RNA ladder standard (GibcoBRL) was used to estimate the length of the RNAs. Probes were synthesized using a random priming kit with 32 P-dATP as the label (Boehringer Mannheim, Indianapolis, IN). Northern analysis was done using the 3' UTR of each full length clone and the RT-PCR clones as templates for probe synthesis. As a loading control, RNA blots were stripped and re-probed at a reduced hybridization and washing stringency using a soybean 26S rDNA fragment (Turano et al., 1997).

For all hybridizations, 32 P(dATP)-labeled probe was diluted to 1-2 x 106 dpm/mL. The complete disclosures of the above references are fully incorporated herein by reference.

Sequence Analysis Sequencing was done at the Iowa State University Sequencing Facility (Ames, IA) using a PCR-based dideoxynucleotide terminator protocol and an ABI automated sequencer (Applied Biosystems, Foster City, CA). The sequencing of both cDNA insert strands was done by primer walking.

Nucleotide and deduced amino acid sequence comparisons against the databases were done using BLAST searches (Altschul et al., 1990). Sequence data were analyzed and aligned using DNA Strider 1.2 (Marck, 1988) and MacDNAsis (Hitachi, San Bruno, CA) software. The complete disclosures of the above references are fully incorporated herein by reference.

WOf OI.AC/.A PCT[US99/12697 6T 77/V..V 29 Northern Blot Analysis Tissue Specific Expression Northern blot analysis was done to reveal which, if any, of the Pgalactosidase genes had a fruit-specific expression pattern. With the exception of TBG2, transcripts of all clones were detected in non-fruit tissues (Fig. 4).

Transcripts of TBG 1, 4, 5 and 6 were detected in all the tissues tested. TBG3 transcript was detected at low levels in root and stem tissues, while TBG7 transcript was detected in flower and stem tissues.

Temporal expression pattern in fruit The temporal expression pattern of the seven genes in fruit tissue was examined using RNA extracted from all fruit tissues except seeds. Transcripts for all seven genes were detected during some stage of fruit development (Fig.

TBGI and 3 had similar expression patterns and their transcripts were detected throughout the breaker to over-ripe stages. TBG2 had a unique expression pattern and its transcript was detected at a constant level from dpp to the over ripe stage. TBG4 expression pattern was similar to TBG1 and 3, but differed in that the transcript level was significantly higher at the turning stage. TBG5 had a similar expression pattern to TBG4 during the ripening stages of development, however TBG5 transcript was also detected throughout all the earlier stages of fruit development. TBG6 had an interesting expression pattern and its transcript was only detected at high levels in all pre-ripening stages tested. TBG7 also had a unique expression pattern and its transcript was detected at very low levels throughout all the stages tested, and at moderate levels at 10 dpp and the over-ripe stage.

PrT/US99/12697 fl// t f{lfC*LA wVj yyo'130o. Spatial expression pattern in fruit Northern blot analysis was also done to determine transcript accumulation in various fruit tissues. Since there were temporal differences in the expression patterns of the TBG genes both the mature green and turning fruit stages were used for RNA extractions (Fig. Both TBG2 and TBG6 transcripts were detected in all mature green fruit tissues tested. TBG7 transcript was present in all fruit tissues tested except for locules. Both TBG1 and TBG4 transcripts were detected in RNA samples extracted from all turning stage fruit tissues. TBG4 transcript was notably more abundant in the peel. TBG3 and TBG5 expression patterns were unique and their transcripts were detected in all tissues except the outer pericarp and locular respectively.

Expression in normal versus mutant fruit In order to better understand the potential roles of the TBG products and transcriptional regulatory mechanisms, northern analysis was performed using fruit tissue from the ripening mutants rin, nor and Nr. This analysis was important because it might give clues for preliminary determination of any potential ripening and/or softening role any of the TBGs might possess.

The results of mutant fruit Northern analysis suggested that the transcriptional regulation of TBG1, 2, 3, 5 and 7 was unaffected in mutant fruit tissue and that their transcripts were present in a normal chronological (dpp) pattern (Fig. The abundance of TBG4 and 6 transcripts were however different in the mutant fruit. TBG4 transcript was not detected in fruit tissue of N' and was detected at much lower levels in rin and nor than wild type fruit WO 99/64564 PCT/US99/12697 31 tissues. Normally TBG6 transcripts are detectable at high levels throughout the early stages of fruit development but are not detectable after the mature green stage (40-42 dpp). TBG6 transcripts persisted even to 50 dpp in fruit of all three mutants.

Transcriptional regulation by ethylene The northern analysis done using mutant and wild type fruit suggested that TBG4 expression might be up-regulated by ethylene and that TBG6 expression might be down-regulated by ethylene. In order to evaluate this hypothesis mature green fruit were harvested and subjected to a continuous flow of 10 ppm ethylene mixed in air. Control and ethylene-treated fruit were used for RNA extractions at 1, 2, 12 and 24 hours. The results of this experiment confirmed the findings from the mutant fruit northern analysis. As expected, the presence and abundance of TBG 1, 2, 3, 5 and 7 transcripts was essentially unaffected in mature green tissues subjected to exogenous ethylene treatment (Fig. However, TBG4 transcript abundance was increased in mature green tissues in the presence of ethylene. From the data presented it was unclear whether TBG6 transcript abundance was reduced by exogenous ethylene treatment since its transcript level was normally reduced at this stage of fruit development.

Enzyme activity In order to determine the role of the TBG encoded products we initiated experiments to express the cDNA encoded enzymes using heterologous expression systems. Several E. coli expression systems were WO 99/64564 pCT/US99/12697 32 tested, but the yield of product was very low due to toxicity See the example below). Therefore we used a yeast expression system which secretes a mature amino-terminal-FLAG fusion protein into the culture medium. The TBG4 cDNA was tested first and resulted in the production of approximately 1 mg TBG4 active protein per 50 mis culture. TBG4 was used first because the cDNA codes for the enzyme -galactosidase I which was purified from tomato fruit and has been characterized in some detail (Carey et al 1995, Smith et al 1998). Therefore we could compare the activity of the heterologous system-expressed protein to the native enzyme purified from tomato. The TBG4 protein was successfully affinity purified using an anti-FLAG affinity resin (Figure 9).

The affinity-purified TBG4 enzyme was shown to have galactosidase activity by virtue of its ability to hydrolyze the synthetic substrate p-nitrophenyl-P-D-galactopyranoside (Smith et al. 1998). The enzyme can cleave galactosyl residues from a variety of cell wall substrates and therefore has exo-galactanase activity (Table II). The remaining fulllength cDNA clones are currently being tested for successful expression of active enzyme. Preliminary results have shown that TBGI codes for an enzyme which also has both 3-D-galactosidase and exo-galactanase activity (Table III).

WO 99/64564 PCT/US99/12697 Table III. Cell wall degrading activity of TBG4 and TBG1 expressed in yeast. Removal of galactosyl residues from chelator soluble (CSP) and alkali soluble (ASP) pectin and hemicellulosic (HCF) cell wall fractions purified from tomato fruit.

gg galactose released enzyme substrate boiled live aTBG4 CSP 0 ASP 0 14.5 HCF 0 4 bTBG1 ASP 0 1.2 2 mg substrate; 4 hours at 37°C a.005 units enzyme/rx 0 0 0 5 units enzyme/rx pZBG2-1-4 Codes for a p-Galactosidase The TBG4 ORF was cloned in-frame into the repressible/inducible bacterial expression vector pFLAG-CTC. The host strain XL1-Blue MR is a mutant strain containing no endogenous p-galactosidase activity nor otcomplementation. Induction of gene transcription by (IPTG) caused the immediate cessation of E. coli growth at 30 to 37°C. However, induction at 20'C did allow for some limited E. coli growth. When clones containing the pZBG2-1-4 4 ORF were grown at 20 0 C and induced with IPTG, the cells slowly turned blue after 36 hrs growth in medium containing the 3galactosidase substrate X-GAL, (Figure 10). If not induced with IPTG, no blue color was seen, even after extended growth in media containing X-GAL.

As an additional negative control, clones consisting of XL1-Blue MR transformed with the FLAG vector alone never showed any p-galactosidase activity with or wilhout PTG-induction, even after 7-days growth (PFi WO 99/64564 PCT/US99/12697 34 As a positive control for maximal p-galactosidase (derived from E. coli Pgalactosidase) activity the cloning vector pGEM was transformed into the host strain DH5c and the results are also shown in Figure 10. Figure 10 shows the detection of p-galactosidase activity from pZBG2-1-4 expression in E. coli.

Cells were harvested and extracts were prepared every 12 hours and the A 61 measured. Cultures were grown with the addition of the chromogenic substrate X-GAL (open symbols) or X-GAL and the transcriptional inducer IPTG (closed symbols) in the medium. The vector used as a positive control for E.

coli p-galactosidase activity was pGEM and the vector used as a negative control and for expression was pFLAG-CTC either without or containing the pZBG2-l-4 ORF Effects on Plant Tissue Texture To further demonstrate the function of TBG4 encoded p-galactosidase 1 the following experiments were carried out.

Fruit from tomato plants containing antisense constructs to suppress TBG4 mRNA were up to 40% firmer [compare means of parental line #1 with antisense line #2 in Figures 11A 11E(1-4)] than fruit from the parental line.

Among the transformants the line with the firmest fruit also had the lowest overall levels of TBG4 mRNA (Figure 12A,B). This correlation suggests that a reduction in TBG4 mRNA is associated with increased fruit firmness. Firmer fruit might result in less shipping damage less loss due to damage and ability to harvest at later stage resulting in better flavor at market longer WO 99/64564 PCT/US99/12697 shelf life for both market and consumer. better quality fruit for fresh slice market; fruit cut better at the pink/red stage when firmer.

Methods To determine the function of TBG4 encoded P-galactosidase II, antisense constructs were made using the constitutively expressed 35S CaMV promoter to express TBG4 antisense RNA (Figure 13). Constructs were moved into tomato using Agrobacterium-mediated transformation. Four tomato cultivars have been transformed in order to evaluate the effect of TBG4 suppression on processing tomato (cv 'UC82b') fruit paste quality and three fresh pick cultivars. Of the fresh pick cultivars one is a soft fruit large cherry tomato (cv 'Ailsa Craig'), the second is a soft fruit old breeding line (cv 'Rutgers') and the third is a recently developed somewhat firm cultivar 'New Rutgers'.

Among the lines where TBG4 mRNA is suppressed we expect to observe an increase in firmness and paste viscosity.

Texture Although this project is nearly finished the complete biochemical and molecular analysis is not finished. The preliminary results on the analysis of the 'New Rutgers' cultivar is presented in Figures 11A E(1-4) and 12A,B. In this example a fresh pick cultivar called 'New Rutgers' was used. Plants of the purchased seed were grown and allowed to self and the resulting seed was used as the parental control (line Seven independent transformed plants (lines 2-8) containing TBG4 antisense constructs were grown and allowed to self. Transformation (T-DNA insertion) was confirmed by southern analysis WO 99/64564 PCT/US99/12697 36 (data not shown). From each transformed line, five plants were grown along with 10 parental line plants. Fruit were tagged at the breaker stage 1 s t onset of color change) and were harvested at breaker plus 7 days. Data were taken using 15-20 fruit from each line. Each type of texture measurement was done twice for each fruit and fruit were subjected to 4 types of texture measurements using a Stable Micro System's TA-XT2i texture analyzer. The 4 measurements were; 1, 2-inch flat plate compression to 3 mm (Figure 1A), 2, 4 mm spherical indenter compression to 3 mm (Figure 1 IB), 3, 4 mm cylindrical indenter compression to 3 mm (Figure 11C) and 4, 4 mm cylindrical indenter puncture to 10 mm (Figure 1 ID). The summary of this data is shown in Figure 11E(l-4). In Figures 11A -E line 1 was the parental line and lines 2-8 each represent an independent transformant containing one T-DNA copy of the TBG4 antisense construct. Statistical analysis (Duncans and Scheff6) of the data revealed that fruit from the transformed lines 3, 7 and 8 were not significantly different from the parental line but that transformed lines 2, 4, and 6 were significantly firmer than the parental fruit. Most noteworthy is that fruit from transformed line 2 had fruit with a mean firmness that was firmer than that of the parental line (Figures 1 1A-D).

Northern Blot Analysis We are currently investigating any changes in the biochemical composition of fruit where TBG4 mRNA levels have been suppressed. These experiments are designed to show a link between increased fruit firmness and TBG4 mRNA suppression, TBG4 encoded enzyme activity suppression, WO 99/64564 PCT/US99/12697 37 possible cell wall modification increased galactosyl residue content) and a decrease in free galactose levels during fruit ripening.

These experiments are not complete, however some preliminary Northern blot experiments were done and the data is shown in Figure 12A,B.

There is no parental or azygous control fruit RNA shown in Figure 12A,B because these plants were the last to grow and RNA extractions are just being done now. As a comparison of normal fruit TBG4 mRNA levels refer to Figure 5 above. The data from Figure 5 showed that TBG4 mRNA levels are low at the mature green stage, peak at the turning stage and are reduced at the red stage. All the lines except for 2 and 3 expressed antisense TBG4 mRNA (Figure 12A,B). The antisense transcripts appear as two bands, smaller in length than the endogenous mRNA. The two bands probably resulted from 1, the expected transcriptional stop signal provided by the NOS-terminator and 2, a cryptic transcriptional stop signal in the antisense TBG4 cDNA. The most notable result was in line 2 where no TBG4 mRNA was detected at the turning stage. Line 2 also had the firmest red fruit (see Figure 11A The absence of detectable TBG4 mRNA probably was the result of cosupression of both the endogenous and antisense mRNAs. When compared to earlier blots (e.g.

Figure all of the lines appeared to have an overall reduced level of TBG4 mRNA, but it is impossible to assign numbers to this statement without the parental and azygous control RNA on the same Northern blot.

The specification discloses that p-galactosidase II polypeptide is involved in the degradation of cell wall pectin during fruit ripening. In the present invention, the role of galactosidases in tomato during fruit ripening and softening and the description of the cloning of a p-galactosidase cDNA clone WO 99/64564 PCT/US99/12697 38 that codes for a p(l-4)galactan degrading enzyme, which is expressed in ripening tomato fruit tissues, has been shown.

The present work indicates that pZBG2-1-4 is a cDNA derived from the transcript of the TBG4 gene which codes for p-galactosidase II for the following reasons: First, the deduced amino acid sequence of the highly conserved aminoterminal portion of the expected mature pZBG2-1-4 translation product matches almost exactly (28 of 30 amino acids) with the amino-terminal sequence of p-galactosidase II as purified by Carey et al. (1995) and designated TOMAA. Importantly, the two amino acids (KY) in the 3galactosidase 1I sequence (TOMAA), that do not match the pZBG2-1-4 deduced amino acid sequence of the present invention are believed to be incorrect since all plant p-galactosidase sequences in the database and four additional P-galactosidase-related cDNAs that were identified from tomato all match or have conserved substitutions with the deduced amino acid sequence of pZBG2-1-4 at these same two amino acid (ST) positions (Figure 3).

Second, the transcript detected by pZBG2-1-4 is present in normal ripening fruit at the same time that p-galactosidase II activity was detected (Figure 5; Carey et al., 1995). Moreover, little or no transcript was detected in fruit at 45 and 50 dpa from the mutants nor, rin and Nr (Figure This observation also coincides with the data presented by Carey et al. (1995) that p-galactosidase II activity remained at levels equal to mature green fruit and did not rise in fruit 45-65 dpa from nor or rin plants. Interestingly, Carrington and Pressey (1996) have _reported that p-gaiactosidase II activity was only WO 99/64564 PCT/US99/12697 39 detected in 'Rutgers' fruit after the turning stage of ripeness. The Northern data in the present invention indicates that maximum p-galactosidase II activity occurs only after the turning stage, assuming mRNA levels predict extractable enzyme activity (Figure Third, the apparent molecular weight of 77.9 kD and pi of 8.9 for the mature protein predicted from the pZBG2-1-4 sequence is similar to that determined for p-galactosidase II., Pressey (1983), estimated a molecular weight of 62 kD by gel-filtration column chromatography and a pi of 7.8 by isoelectric focusing, while Carey et al. (1995) estimated a molecular weight of 75 kD by SDS-PAGE and a pi of 9.8 by isoelectric focusing.

Fourth, enzyme produced from pZBG2-1-4 ORF using a heterologous yeast expression system has both p-galactosidase activity and exogalactinase activity.

WO 99/64564 PCT/US99/12697 LITERATURE CITED Altschul SF, Gish W, Miller W, Meyers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403-410 Ausubel I. Brent R, Kingston R, Moore D, Seidman J, Smith J, Struhl K, eds. (1987) Current Protocols in Molecular Biology. John Wiley and Sons. New York Carey AT, Holt K. Picard S, Wilde R, Tucker GA, Bird CR, Schuch W, Seymour GB (1995) Tomato P-D-galactanase. Isolation, changes during ripening in normal and mutant tomato fruit, and characterization of a related clone. Plant Physiol 108:1099-1107 Carrington CM, Pressey R (1996) P-galactosidase II activity in relation to changes in cell wall galactosyl composition during tomato ripening. J Amer Soc Hort Sci 121:132-136 Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81:1991-1995 DeVeau EJ, Gross KC, Huber DJ, Watada AE (1993) Degradation and solubilization of pectin by P-galactosidases purified from avocado mesocarp. Physiol Plant 87:279-285 Fischer RL, Bennett AB (1991) Role of cell wall hydrolases in fruit ripening.

Annu Rev Plant Physiol Plant Mol Bio 42:675-703 Giovannoni JJ, DellaPenna D, Bennett AB, Fischer RL (1989) Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening.

Plant Cell 1:53-63 WO 99/64564 PCT/US99/12697 41 Golden KD, John MA, Kean EA (1993) P-Galactosidase from Coffea arabica and its role in fruit ripening. Phytochemistry 34:355-360 Gross KC (1984) Fractionation and partial characterization of cell walls from normal and non-ripening mutant tomato fruit. Physiol Plant 62:25-32 Gross KC, Sams CE (1984) Changes in cell wall neutral sugar composition during fruit ripening: A species survey. Phytochemistry 23:2457-2461 Gross KC, Wallner SJ (1979) Degradation of cell wall polysaccharides during tomato fruit ripening. Plant Physiol 63:117-121 Hall LN, Tucker GA, Smith CJS, Watson CF, Seymour GB, Bundick Y, Boniwell JM, Fletcher JD, Ray JA, Schuch W, Bird CR, Grierson D.

(1993) Antisense inhibition of pectin esterase gene expression in transgenic tomatoes. Plant J 3:121-129 Huber DJ (1983) The role of cell wall hydrolases in fruit softening. Hort Rev 5:169-219 Kang IK, Suh SG, Gross KC, Byun JK (1994) N-terminal amino acid sequence of persimmon fruit 3-galactosidase. Plant Physiol 105: 975-979 Kim J, Gross KC, Solomos T (1991) Galactose metabolism and ethylene production during development and ripening of tomato fruit. Postharv Biol Technol 1:67-80 Marck C (1988) DNA Strider: a program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers.

Nucleic Acids Res 16:1829-1836 WO 99/64564 PCT/US99/12697 42 Mitcham EJ, Gross KC, Ng TJ (1989) Tomato fruit cell wall synthesis during development and senescence. In vivo radiolabeling of cell wall fractions using 4 C]sucrose. Plant Physiol 89:477-481 Nakai K, Kanehisa M (1992) A knowledge base for predicting protein localization sites in eukaryotic cells. Genomics 14:897-911 Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 10:1-6 Pressey R (1983) P-Galactosidases in ripening tomatoes. Plant Physiol 71:132-135 Ross GS, Redgwell RJ, MacRae EA (1993) Kiwifruit p-galactosidase: isolation and activity against specific fruit cell-wall polysaccharides.

Planta 189:499-506 Ross GS, Wegrzyn T, MacRae EA, Redgwell RJ, (1994) Apple Pgalactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone. Plant Physiol 106:521-528 Seymour GB, Gross KC (1996) Cell wall disassembly and fruit softening.

Postharvest News Info 7:45N-52N Smith CJS, Watson CFS, Ray J, Bird CR, Morris PC, Schuch W, Grierson D (1988) Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334:724-726 Smith DL, Fedoroff NV (1995) LRP1, a gene expressed in lateral and adventitious root primordia of Arabidopsis. Plant Cell 7: 735-745 WO 99/64564 PCT/US99/12697 43 Smith DL, Starrett DA and Gross KC (1998) A gene coding for tomato fruit P-galatosidase II is expressed during fruit ripening. Plant Physiol. 117: 417-423 Tieman DM, Harriman RW, Ramamohan G, Handa AK (1992) An antisense pectin methylesterase gene alters pectin chemistry and soluble solids in tomato fruit. Plant Cell 4:667-679 Tigchelaar EC, McGlasson WB, Buescher RW (1978) Genetic regulation of tomato fruit ripening. HortScience 13:508-513 Turano FJ, Thakkar SS, Fang T, Weisemann JM (1997) Characterization and expression of NAD(H) dependent glutamate dehydrogenase genes in Arabidopsis thaliana. Plant Physiol 113: 1329-1341 Verwoerd TC, Dekker BMM, Hoekema A (1989) A small-scale procedure for the rapid isolation of plant RNAs. Nuc Acids Res 17: 2362 Wegrzyn TF, MacRae EA (1992) Pectinesterase, polygalacturonase, and 3galactosidase during softening of ethylene-treated kiwifruit. HortScience 27:900-902

Claims (15)

1. An isolated nucleic acid molecule comprising a polynucleotide encoding a p- galactosidase II polypeptide or the complement thereof and having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: a nucleotide sequence encoding the p-galactosidase II polypeptide having the complete amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, and designated TBG2, TBG4, TBG5, TBG6 and TBG7, respectively as shown in Figure 2 or as encoded by the cDNA clone selected from the group consisting of cDNA clones contained in Gen Bank Accession No. AF154420, AF154421, AF020390, AF154423, AF154424 and AF154422; a nucleotide sequence encoding the mature P-galactosidase II polypeptide having the amino acid sequence at about positions 24-724 selected from the group consisting of, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, 15 and designated TBG2, TBG4, TBG5, TBG6 and TBG7, respectively as shown in Figure 2 or S: as encoded by the cDNA clone selected from the group consisting of cDNA clones contained in Gen Bank Accession No. AF154420, AF020390, AF154423, AF154424 and AF154422; .and a nucleotide sequence complementary to any of the nucleotide sequences in (a) or above.

2. The nucleic acid molecule of claim 1 wherein said polynucleotide has the complete nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 as shown in Figure 2. *oooo *ooo

3. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence in Figure 2 (SEQ ID NO: 4) encoding the P-galactosidase II polypeptide having the amino acid sequence designated TBG4 in Figure 2.

4. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence in Figure 2 (SEQ ID NO: 4) encoding the mature polypeptide having the amino acid sequence from about 24 to about 724 in the amino acid sequence designated TBG4 in Figure 2. The nucleic acid molecule of claim 1 wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in Gen Bank Accession No. AF154420. 15 6. The nucleic acid molecule of claim 1 wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in Gen Bank Accession No. AF154421.

7. The nucleic acid molecule of claim 1 wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in Gen Bank Accession No. AF020390. 58 7. The nucleic acid molecule of claim 1 wherein said polynucleotide has the 2 complete nucleotide sequence of the cDNA clone contained in Gen Bank Accession No. 2 AF154423.

27-10-04; 16:06 PIZZ YS :61732218077 7/ 12 46 9. The nucleic acid molecule of claim 1 wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in Gen Bank Accession No. AF 154424. The nucleic acid molecule of claim 1 wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in Gen Bank Accession No. AF154422. 11. An isolated nucleic acid molecule comprising a polynucleotide encoding a P- galactosidase II polypeptide or complement thereof which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to a nucleotide sequence in or of claim I wherein said polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. 12. An isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a 3- galactosidase II polypeptide having an amino acid sequence in or of claim 1, wherein said polypeptide displays 20 P- galactosidase II activity. 13. A method for making a recombinant vector comprising inserting an isolated nucleic acid molecule of claim 1 into a vector. 14. A recombinant vector produced by the method of claim 13. A method of making a recombinant host cell comprising introducing the S* recombinant vector of claim 14 into a host cell. 16. A recombinant host cell produced by the method of claim COMS ID No: SBMI-00973412 Received by IP Australia: Time 15:56 Date 2004-10-27 17. A recombinant method for producing p-galactosidase II polypeptide, comprising culturing the recombinant host cell of claim 16 under conditions such that said polypeptide is expressed and recovering said polypeptide. 18. An isolated p-galactosidase II polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: a) amino acid sequence at about positions 24-724 selected from the group consisting of sequences SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, and designated TBG2, TBG4, TBG5, TBG6 and TBG7, respectively as shown in Figure 2; and b) amino acid sequence as encoded by the cDNA clone selected from the group consisting of cDNA clones contained in Gen Bank Accession No. AF154420, AF020390, AF154423, AF154424 and AF154422. S 19. An isolated polypeptide comprising an epitope-bearing portion of the 3- galactosidase II protein. 20. An isolated antibody that binds specifically to a P-galactosidase II polypeptide of claim 19. 2 7 -10 04:16:'0 6 :P17 7Z EYS :6173 22 18 07 7 8/12 48 21. An isolated nucleic acid molecule comprising a polynucleotide encoding a 0 galactosidase II polypeptide or the complement thereof and having a nucleotide sequence at least 95% identical to a sequence selected fr'om, the group consisting of. a nucleotide sequence encoding the P-galactosidase II polypeptide having the complete amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, and designated TBG2, TBG4, TBG5, TBG6 and TB07, respectively as shown in Figure 3 or as encoded by the cDNA clone selected from the group consisting of cDNA clones contained in Gen Bank Accession No. AF 154420, AF020390, AF 154423, Af 154424 and AF 154422; a nucleotide sequence encoding the mature P-ga~actosidase 1I polypeptide having the amino acid sequence at about positions 24-724 selected from the group consisting of sequences SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, and designated TBG2. TBG4, TBG5, TBG6 and TBG7, respectively as shown in Figure 3 or as encoded by the cDNA clone selected from the group consisting of cDNA clones contained in Gen Bank Accession No. AF154420, AF020390, AF154423, AF154424 and AF 154422; and a nucleotide sequence complementary to any of the nucleotide sequences in (a) or above.. 20 22. The nucleic acid molecule of claim 21 wherein said polynucleotide has a comnplete nucleotide sequence in Figure 2 selected from the group consisting of SEQ ID NOs: 2 and 5-7. COMS ID No: SBMI-00973412 Received by IP Australia: Time 15:56 Date 2004-10-27 23. The nucleic acid molecule of claim 21 wherein said polynucleotide has a nucleotide sequence in Figure 2 selected from the group consisting of SEQ ID NOs: 2-3 and 5-7 encoding the p-galactosidase polypeptide having the complete amino acid sequence designated TBG2-3 and 5-7, respectively. 24. The nucleic acid molecule of claim 21 wherein said polynucleotide has the nucleotide sequence in Figure 2 selected from the group consisting of SEQ ID NOs: 2-3 and 5-7 encoding the mature polypeptide having the amino acid sequence designated TBG2-3 and 5-7, respectively. The nucleic acid molecule of claim 21 wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in an Gen Bank Accession No. selected from the group consisting of AF154420, AF020390, AF154423, AF154424 and 15 AF154422. 26. A method of modifying cell wall metabolism in a plant which comprises S: transforming said plant with a DNA construct adapted to modify the activity of a P- galactosidase, growing said plant or its descendent and selecting a plant having modified cell wall characteristics, said construct comprising a transcriptional initiation region operative in plants operably linked to a DNA sequence according to any one of claims 1 to 25, encoding at least one P-galactosidase. 25 27. A plant cell transformed with a nucleic acid molecule as claimed in any one of claims I to

28. A plant derived from a plant cell as claimed in claim 27.

29. A plant seed derived from a plant as claimed in claim 28. A method for modifying p-galactosidase gene expression in a plant comprising transforming said plant with a nucleic acid molecule as claimed in any one of claims 1 to S7 -10 04 :16 06 P Z :_Y 61732218077 9/ 12 growing the transformed plant and selecting a plant having modified p-galactosidase gene expression when compared with an untransformed plant.

31. A method of claim 26, wherein said plant is a fruiting plant.

32. A method of modifying ripening in a fruit which comprises transforming a fruiting plant with a DNA construct adapted to modify the activity of a p-galactosidase, growing said plant or its descendent and selecting a plant having modified cell wall characteristics, said construct comprising a transcriptional initiation region operative in plants operably linked to a DNA sequence according to any one of claims 1 to 25, encoding at least one P-galactosidase.

33. An isolated nucleic acid molecule comprising a polynucleotide encoding B- galactosidase II polypeptide or complement thereof substantially as herein described in the Example with reference to the Figures.

34. A method for making a recombinant vector substantially as herein described in the Example with reference to the Figures. S:

35. A recombinant vector substantially as herein described in the Example with reference to the Figures.

36. A plant transformed with a nucleic acid molecule substantially as herein 25 described in the Example with reference to the Figures.

37. A method of modifying cell wall metabolism in a plant substantially as herein described in the Example with reference to the Figures. DATED THIS TWENTY-SEVENTH DAY OF OCTOBER 2004 UNITED STATES DEPARTMENT OF AGRICULTURE BY PIZZEYS PATENT AND TRADE MARK ATTORNEYS COMS ID No: SBMI-00973412 Received by IP Australia: Time 15:56 Date 2004-10-27