WO2007139385A1 - Methods for modulating potato tuber cooking type and plant tissue texture - Google Patents

Abstract

The invention relates to the field of plant biotechnology and plant breeding. Provided are methods for modulating the texture properties of plant tissues, especially the cooking type of root and tuber vegetables, such as potato tubers, and fruits. Also provided are nucleic acid and amino acid sequences useful for marker assisted selection and for making transgenic plants having altered texture characteristics.

Description

Methods for modulating potato tuber cooking type and plant tissue texture

FIELD OF THE INVENTION

The invention relates to the field of plant biotechnology and plant breeding. Provided are methods for modulating the texture properties of plant tissues and organs, especially after heat treatment, such as cooking. Novel cell wall proteins and variants thereof are provided, as well as nucleic acid sequences encoding these. Especially the cooking type of potato tubers can be altered by the methods of the invention, for example from

'mealy' to 'firm', or vice versa. Similarly, the texture of fruit and vegetables (especially root and tuber vegetables) can be altered using the proteins and nucleic acid sequences according to the invention.

BACKGROUND OF THE INVENTION

Differential gene expression within a population can be considered as a quantitative trait that can result in the mapping of gene expression as a proper QTL or so-called eQTL (Schadt et al, 2003, Nature 422, 297-302). The combination of expression profiling and genetics has been referred to as 'genetical genomics' and is expected to greatly advance our capabilities to resolve metabolic, regulatory and developmental pathways (Jansen and Nap, 2001, Trends Genet 17, 388-391). The use of microarray technology for accurately scoring of differential gene expression within large populations has already resulted in the identification of novel candidate genes underlying specific traits of interest (Brem et al., 2002, Science 296, 752-755; Wayne and Mclntyre, 2002, Proc Natl Acad Sci U S A 99, 14903-14906; Schadt et al., 2003, supra; Kirst et al., 2004, Plant Physiol 135, 2368-237.). Efficient analysis of the entire transcriptome is however still limited to organisms of which the genome sequence has been largely determined or for which comprehensive EST databases are available.

In a previous study (Kloosterman et al., 2005, Plant Biotechnology Journal 3, 505-519), the inventors have developed a dedicated cDNA-microarray, containing around 2000 elements specifically designed for studying gene expression during potato tuber development and tuber quality traits. They now implement a novel pooling strategy, or bulk segregant analysis approach (BSA; Michelmore et al., 1991, Proc Natl Acad Sci U S A 88, 9828-9832), for studying gene expression in a diploid potato population. Thereby they identified genes encoding novel proteins (termed herein "StTLRP" proteins) specifically involved in the determination of the textural changes in plant tissues, especially in potato tubers after steam cooking (i.e. cooking type). These genes and proteins can be used as described herein for modulating texture characteristics of plants, plant tissues and organs, such as modified undergrounds stems (tubers, corm, rhizomes), true roots, tuberous roots or (fleshy) fruit.

Texture is an important quality trait of potato tubers and many other fruits and root vegetables. For example, the texture of cooked potatoes is an economically important quality aspect and is generally characterized as the differences between 'mealy' and 'non-mealy/firm' tubers. A mealy tuber is one which, while it retains its form on cooking, may readily be broken down with a fork to give a dry crumbly mash through separation of individual cells (Burton, 1966, The Potato, A survey of its history and of factors influencing its yield, nutritive value, quality and storage, Second edition Edn: H. Veenman & Zonen N.V. Wageningen, Holland). A firm tuber on the other hand, does not break down easily in comparison to mealy tubers but when forced is accompanied by a significant amount of cell breakage (Burton, 1966, supra; van Marie et al, 1992, Food Structure 11, 209-216).

Textural changes occurring during cooking are mainly associated with cell wall and middle lamella structural components and the gelatinization characteristics of starch (van Marie, 1997, Characterization of changes in potato tissue during cooking in relation to texture development. PhD thesis. Wageningen: Wageningen University; Alvarez and Canet, 1998, Z Lebensm Unters Forsch A 207, 55-65). One of the parameters to categorize cooking behaviour is sloughing: the loosening of the outer layers of the cooked potato. Sloughing is determined by the amount of intercellular adhesion within cooked potato tissue (Jarvis and Duncan, 1992, Potato Res. 35, 83-91; van Marie et al., 1992, supra) and can be easily assessed (and optionally scored) visually after cooking. Van Marie et al. (1994, Potato Res. 37, 183-195), showed that both cell sloughing and the release of pectic materials were higher for the mealy cooking potato cultivar Irene than for the non-mealy/firm cooking cultivar Nicola. Furthermore, a difference in cell wall thickness was observed, accompanied by a difference in the degree of middle lamella breakdown during cooking which was higher in cultivar Irene in comparison to cultivar Nicola (van Marie et al., 1997, J Agric Food Chem 45, 50-58). Studies on the assembly process of cell walls have provided insight in the architecture of the primary cell wall and led to the identification of a number of genes and structural proteins that are associated with modifying cell wall characteristics such as permeability, expansion, stress relaxation and mechanical strength (Showalter, 1993, Plant Cell 5, 9-23; Cosgrove, 1997, Annu Rev Cell Dev Biol 13, 171-201). However, the genetic components and genes involved in potato tuber cell wall characteristics in relation to the differences in cooking type have not been fully understood and there is clear lack of high-quality suitable genes.

Similarly, plant varieties differ in the texture of the harvested parts, such as the fruits and root vegetables. For example, (as in potato) mealy apple varieties and firm apple varieties exist. In addition post harvest textural changes of fruit, such as members of the genus Prunus and Malus, as well as other fruit, such as tomatoes (Solarium lycopersicum syn. Lycopersicon esculentum), result in loss of quality and can limit shelf life and storage time. For example, low temperature storage may result in "mealiness" (or woolliness), characterized by loss of juiciness and a mealy texture. Mealiness is generally evaluated by mechanical, physical or sensory means (sensory panels) and is considered an undesired characteristic by consumers.

It is, therefore, an object of the invention to provide new methods, genes and proteins for modulating cell wall and texture characteristics of plant tissues and organs and, especially, to provide means for increasing firmness and/or reducing mealiness. In certain embodiments also methods and means for reducing firmness (increasing softness) and/or enhancing mealiness are provided.

GENERAL DEFINITIONS

"Root vegetables" or "root and tuber vegetables" is a generic term used herein to refer to plant storage organs growing underground, which are harvested and consumed by humans and animals. This term encompasses anatomically and developmentally different tissue types, such as "true roots" (e.g. turnip roots, carrot, sugar beet, etc.), "tuberous roots" (e.g. sweet potato, cassava, etc.) and various modified underground stems. Modified stems can be subdivided into "corm" (e.g. taro), "Rhizomes" and "tubers" (e.g. potato, yam, etc.).

The term "nucleic acid sequence" (or nucleic acid molecule) refers to a DNA or RNA molecule in single or double stranded form, particularly a DNA encoding a protein or protein fragment according to the invention. An "isolated nucleic acid sequence" refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g. the nucleic acid sequence in a bacterial host cell or in the plant nuclear or plastid genome. The terms "protein" or "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 dimensional structure or origin. A "fragment" or "portion" of a StTLRP protein may thus still be referred to as a "protein". An "isolated protein" is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell. The term "gene" means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene may thus comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3 'non-translated sequence comprising e.g. transcription termination sites.

The term "allele(s)" means any of one or more alternative forms of a gene at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus. In a diploid cell of an organism, alleles of a given gene are located at a specific location, or locus (loci plural) on a chromosome. One allele is present on each chromosome of the pair of homologous chromosomes. A diploid plant species may comprise a large number of different alleles at a particular locus. These may be identical alleles of the gene (homozygous) or two different alleles (heterozygous). The term "locus" (loci plural) means a specific place or places or a site on a chromosome where for example a gene or genetic marker is found.

"Marker assisted selection" refers to the use of molecular marker assays in breeding programs (involving e.g. crossing, selling, backcrossing, etc.), whereby (directly or indirectly) the presence or absence of particular alleles or allele combinations at the StTLRP locus are determined and whereby breeding procedures can be speeded up significantly as no or less phenotypic tests (e.g. tissue texture assays) are required for selection of plants comprising the desired allele / allele combinations. "Molecular marker assay" (or test) refers to a (DNA or RNA based or amino acid based) assay that indicates (directly or indirectly) the presence or absence of a particular allele or allele combination at the StTLRP locus. Preferably it allows one to determine whether a particular allele is homozygous or heterozygous at the locus in any individual plant. A "chimeric gene" (or recombinant gene) refers to any gene, which is not normally found in nature in a species, in particular a gene in which one or more parts of the nucleic acid sequence are present that are not associated with each other in nature. For example the promoter is not associated in nature with part or all of the transcribed region or with another regulatory region. The term "chimeric gene" is understood to include expression constructs in which a promoter or transcription regulatory sequence is operably linked to one or more coding sequences or to an antisense (reverse complement of the sense strand) or inverted repeat sequence (sense and antisense, whereby the RNA transcript forms double stranded RNA upon transcription). "Expression of a gene" refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide (or active peptide fragment) or which is active itself (e.g. in posttranscriptional gene silencing or RNAi). An active protein in certain embodiments refers to a protein having a dominant-negative function due to a repressor domain being present. The coding sequence is preferably in sense-orientation and encodes a desired, biologically active protein or peptide, or an active peptide fragment. In gene silencing approaches, the DNA sequence is preferably present in the form of an antisense DNA or an inverted repeat DNA, comprising a short sequence of the target gene in antisense or in sense and antisense orientation. "Ectopic expression" refers to expression in a tissue in which the gene is normally not expressed. A "transcription regulatory sequence" is herein defined as a nucleic acid sequence that is capable of regulating the rate of transcription of a (coding) sequence operably linked to the transcription regulatory sequence. A transcription regulatory sequence as herein defined will thus comprise all of the sequence elements necessary for initiation of transcription (promoter elements), for maintaining and for regulating transcription, including e.g. attenuators or enhancers. Although mostly the upstream (5') transcription regulatory sequences of a coding sequence are referred to, regulatory sequences found downstream (3') of a coding sequence are also encompassed by this definition. As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically (e.g. by external application of certain compounds) or developmentally regulated. A "tissue specific" or "tissue preferred" promoter is only / mainly active in specific types of tissues or cells.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter, or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame so as to produce a "chimeric protein".

A "chimeric protein" or "hybrid protein" is a protein composed of various protein "domains" (or motifs) which is not found as such in nature but which a joined to form a functional protein, which displays the functionality of the joined domains (for example DNA binding or repression leading to a dominant negative function). A chimeric protein may also be a fusion protein of two or more proteins occurring in nature. The term "domain" as used herein means any part(s) or domain(s) of the protein with a specific structure or function that can be transferred to another protein for providing a new hybrid protein with at least the functional characteristic of the domain. Specific domains can also be used to identify protein members belonging to the StTLRP proteins, such as orthologs from other plant species or allelic variants within the same species. Domains found in StTLRP proteins are the "Cysteine Domain", the "N- terminal domain" (comprising the putative secretion signal peptide), and the "middle domain" (between putative signal peptide and Cysteine Domain), including variants of any of these.

The terms "target peptide" refers to amino acid sequences which target a protein to intracellular organelles such as plastids, preferably chloroplasts, mitochondria, or to the extracellular space (secretion signal peptide). A nucleic acid sequence encoding a target peptide may be fused (in frame) to the nucleic acid sequence encoding the amino terminal end (N-terminal end) of the protein or the nucleic acid sequence encoding an existing (putative) target peptide may be replaced.

A "nucleic acid construct" or "vector" is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology and which is used to deliver exogenous DNA into a host cell. The vector backbone may for example be a binary or superbinary vector (see e.g. US5591616, US2002138879 and WO9506722), a co-integrate vector or a T-DNA vector, as known in the art and as described elsewhere herein, into which a chimeric gene is integrated or, if a suitable transcription regulatory sequence is already present, only a desired nucleic acid sequence (e.g. a coding sequence, an antisense or an inverted repeat sequence) is integrated downstream of the transcription regulatory sequence. Vectors usually comprise further genetic elements to facilitate their use in molecular cloning, such as e.g. selectable markers, multiple cloning sites and the like (see below). A "host cell" or a "recombinant host cell" or "transformed cell" are terms referring to a new individual cell (or organism) arising as a result of at least one nucleic acid molecule, especially comprising a chimeric gene encoding a desired protein or a nucleic acid sequence which upon transcription yields an antisense RNA or an inverted repeat RNA (or hairpin RNA) for silencing of a target gene/gene family, having been introduced into said cell. The host cell is preferably a plant cell or a bacterial cell. The host cell may contain the nucleic acid construct as an extra-chromosomally (episomal) replicating molecule, or more preferably, comprises the chimeric gene integrated in the nuclear or plastid genome of the host cell. The term "selectable marker" is a term familiar to one of ordinary skill in the art and is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing the selectable marker. Selectable marker gene products confer for example antibiotic resistance, or more preferably, herbicide resistance or another selectable trait such as a phenotypic trait (e.g. a change in pigmentation) or a nutritional requirements. The term "reporter" is mainly used to refer to visible markers, such as green fluorescent protein (GFP), eGFP, luciferase, GUS and the like. The term "ortholog" of a gene or protein refers herein to the homologous gene or protein found in another species, which has the same function as the gene or protein, but (usually) diverged in sequence from the time point on when the species harbouring the genes diverged (i.e. the genes evolved from a common ancestor by speciation). Orthologs of the Solanum tuberosum sttlrp genes may thus be identified in other plant species based on both sequence comparisons (e.g. based on percentages sequence identity over the entire sequence or over specific domains) and functional analysis. The terms "homologous" and "heterologous" refer to the relationship between a nucleic acid or amino acid sequence and its host cell or organism, especially in the context of transgenic organisms. A homologous sequence is thus naturally found in the host species (e.g. a potato plant transformed with a potato gene), while a heterologous sequence is not naturally found in the host cell (e.g. a potato plant transformed with a sequence from tomato plants). Depending on the context, the term "homolog" or "homologous" may alternatively refer to sequences which are descendent from a common ancestral sequence (e.g. they may be orthologs).

"Stringent hybridisation conditions" can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe. Typically stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60⁰C. Lowering the salt concentration and/or increasing the temperature increases stringency. Stringent conditions for RNA-DNA hybridisations (Northern blots using a probe of e.g. 100nt) are for example those which include at least one wash in 0.2X SSC at 63°C for 20min, or equivalent conditions. Stringent conditions for DNA-DNA hybridisation (Southern blots using a probe of e.g. lOOnt) are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50⁰C, usually about 55°C, for 20 min, or equivalent conditions. See also Sambrook et al. (1989) and Sambrook and Russell (2001).

"Sequence identity" and "sequence similarity" can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as "substantially identical" or "essentially similar" when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimises the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA. Alternatively percent similarity or identity may be determined by searching against databases such as FASTA, BLAST, etc. In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". It is further understood that, when referring to "sequences" herein, generally the actual physical molecules with a certain sequence of subunits (e.g. amino acids) are referred to.

As used herein, the term "plant" includes the whole plant or any parts or derivatives thereof, such as plant organs (e.g. harvested storage organs, tubers, fruit, leaves, etc.), plant cells, plant protoplasts, plant cell tissue cultures from which whole plants can be regenerated, plant calli, plant cell clumps, and plant cells that are intact in plants, or parts of plants, such as embryos, pollen, ovules, fruit (e.g. harvested tissues or organs), flowers, leaves, seeds, tubers, clonally propagated plants, roots, stems, root tips and the like. Also any developmental stage is included, such as seedlings, cuttings prior or after rooting, etc.

As used herein, the term "variety" or "cultivar" means a plant grouping within a single botanical taxon of the lowest known rank, which can be defined by the expression of the characteristics resulting from a given genotype or combination of genotypes.

DETAILED DESCRIPTION

The present inventors found a new (sub)group of proteins, referred herein to as StTLRP proteins, (and sttlrp nucleic acids encoding these) suitable for modulating texture characteristics of plant cell walls, especially cells (and tissues consisting largely thereof) which lack a rigid (lignifϊed) secondary cell wall. A significant correlation between mRNA expression levels and firmness of potato tubers was found. Potato genop types comprising a specific allele of the sttlrp gene (the sttlrp Δ7 allele) had sttlrp mRNA levels which were much higher (e.g. 64-fold upregulated) than genotypes lacking this allele. These genotypes had on average much firmer tissue after cooking. In contrast, genotypes which lacked the Δ7 allele were mealy after cooking. Therefore, particular high expressing alleles are able to confer a 'firm', 'non-mealy' phenotype to the potato tubers, while low expressing alleles (and thus the absence of high expressing alleles) are capable of conferring a 'mealy' phenotype. How the nucleic acid sequence encoding these new proteins, and both natural alleles and artificial variants thereof, can be used to modulate tissue texture characteristics is described herein below and in the Examples.

Proteins according to the invention

The proteins according to the invention are referred to as "StTLRP" proteins (Solanum tuberosum Tyrosine and Lysine Rich Protein). This name was simply chosen due to amino acid homology with a known tomato and tobacco protein known under the name TLRP and NtTLRP, respectively. This name is not intended to limit the present invention in any way, and no functional relatedness can be inferred from the name. Based on purely the structural similarity, one can refer to the StTLRP proteins as being "family members" of the known tomato and tobacco proteins, but because the present proteins have a different function in vivo it is preferred herein to refer to the StTLRP proteins as a new (sub)group of proteins.

Thus, the two allelic variants of the StTLRP protein shown in SEQ ID NO: 1 (Δ7 allele) and SEQ ID NO: 2 (allele without 7 amino acid deletion in the Cysteine Domain) have some sequence homology to these prior art proteins (see Table below), but their in vivo function is different. The tomato and tobacco TLRP proteins are thought to be involved in strengthening cell walls associated with the vascular tissue (tracheae), especially the xylem, i.e. with cells making a secondary cell wall (see Table 2 in Domingo et al. 1994, The Plant Cell 6: 1035-1047 and Domingo et al. 1999, The Plant Joural 20: 563-570). In contrast, the proteins according to the invention were found to modify texture characteristics of potato tubers, i.e. ground tissue which consist largely of sclerenchyma cells lacking a secondary cell wall. In addition the proteins according to the invention can also be structurally differentiated from the tomato and tobacco proteins, as shown in Table 1.

Table 1 - percentage sequence identity

Pairwise alignments were done using the Program "Needle" of Emboss Win Version 2.10.0, using a Gap opening penalty of 8.0 and a Gap extension penalty of 2.0 and Blosum62.

In accordance with the invention "StTLRP proteins", and "variants" thereof, refer to proteins comprising at least 77% or 78%, preferably at least 79%, 80%, 85%, 88%, 89%, 90%, 92%, 95%, 96%, 98%, 99% or more (100%) amino acid identity to the proteins depicted in SEQ ID NO: 1 and/or SEQ ID NO: 2.

The StTLRP proteins and variants according to the invention are about 87 amino acids long, or shorter, such as 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76 or 75 amino acids. In one embodiment of the invention the putative signal peptide may be deleted or replaced with another signal peptide, without altering the biological activity of the protein. Such variants may thus be shorter (e.g. by up to about 25 amino acids) and comprise at least

70%, 75%, preferably at least 80%, 85%, 88%, 89%, 90%, 92%, 95%, 96%, 98%, 99% or more (100%) amino acid identity to the proteins of SEQ ID NO: 3 and/or 4

(excluding any signal peptides when doing pairwise alignments).

The two variants depicted in SEQ ID NO: 1 and 2 and in Figure 3 have the following characteristics:

SEQ ID NO: 1 (Δ7 protein):

- 78 amino acids, of which 4 cysteines (C), 7 lysines (K) and 7 tyrosines (Y)

- molecular weight 8.7 kDa

- charge 4.5 - isoelectric point 8.3

- putative N-terminal secretion signal peptide amino acids 1-25 having the following sequence: 5' MGSKAIMFLGLFLAIFLMISSEVAA 3'

- middle domain comprising the sequence YHGGGYGK

- Cysteine Domain comprising the sequence CXXCXXXXCC, especially CXXCXKKXCC.

SEQ ID NO: 2: - 85 amino acids, of which 5 cysteines (C), 10 lysines (K) and 9 tyrosines (Y)

- molecular weight 9.5 kDa

- charge 7.5

- isoelectric point 8.9 - putative N-terminal secretion signal peptide amino acids 1-25 having the following sequence: 5' MGSKAIMFLGLFLAIFLMISSEVAA 3'

- middle domain comprising the sequence YHGGGYGK

- Cysteine Domain comprising the sequence C(X)₂ (C)₂ XXXXXX C(X)₃CC

Thus, a protein according to the invention can be identified by the percentage amino acid sequence identity to SEQ ID NO: 1 and/or 2 (and/or to SEQ ID NO: 3 and/or 4) as defined above and preferably further by the presence of a cysteine domain, preferably at the C-terminal end of the protein. A "cysteine domain" refers herein to a domain comprising at least 4 cysteines, having the consensus sequence: C(X)_2-3 (C)_1-2 (X)_4-6 (C)O_-1(X)_0-3CC. This cysteine domain encompasses variants, such as C(X)₂Ci(X)₄ C₀X₀CC (found in SEQ ID NO: 1) and C(X)₂ (C)₂ (X)₆ Ci(X)₃CC (found in SEQ ID NO: 2 and also in the known tomato TLRP protein). X can be any amino acid. It is noted that the cysteine domain of SEQ ID NO: 1 is 10 amino acids in length and comprises 7 amino acid deletions with respect to the domain of SEQ ID NO: 2 (17 amino acids).

Further, a middle domain and/or an N-terminal domain comprising a signal sequence may be present. The amino acid sequence of these domains may thus also encompass variants, for example sequences comprising at least 70, 75, 80, 90, 95, 98% or more amino acid sequence identity to the N-terminal or middle domain or consensus cysteine domain or to the cysteine domain found in SEQ ID NO: 1 or 2.

Optionally one or more amino acids may be added, deleted or replaced in the conserved cysteine domain and/or N-terminal domain and/or middle domain, whereby the biological activity of the protein is not reduced and is preferably increased. Preferred cysteine domains are domains comprising deletions and/or amino acid replacements of 1, 2, 3, 4, 5, 6, 7 or 8 amino acids with respect to the longest consensus cysteine domain, which is 18 amino acids long [C(X)₃ (C)₂ (X)₆ Ci(X)₃CC]. Preferably, at least 4 of the cysteines are retained in these variants.

In one embodiment, the amino acid deletions and/or replacements in the cysteine domain result in a higher expression level compared to nucleic acid sequences encoding the protein of SEQ ID No: 2 and/or the stability (and functionality) of the transcript encoding the protein is increased. Without limiting the invention, it is thought that the nucleic acid sequence encoding amino acid sequences comprising deletions in the consensus cysteine domain (such as the Δ7 variant of SEQ ID NO: 1) confer higher expression, which in turn is correlated with either more functional protein or protein having enhanced stability and cell wall strengthening capability. Proteins comprising the consensus cysteine domain, or a variant cysteine domain such as domains comprising at least 4 cysteines and being at least 10 amino acids in length, are therefore a particularly preferred embodiment of the invention. The functionality of the proteins in modulating tissue texture characteristics can be tested for example by overexpression, as described below.

"Variants" include both artificial variants, made e.g. by recombinant DNA technology or protein synthesis or by mutagenesis methods, as well as natural variants, found in natural plant populations. Variants of the StTLRP proteins include for example proteins having some, e.g. 2, 5, 10, 15 or more, amino acids added, replaced or deleted without significantly changing the protein activity or at least without reducing the activity in vivo when compared to SEQ ID NO: 1 and/or 2. For example conservative amino acid substitutions within the categories basic (e. g. Arg, His, Lys), acidic (e. g. Asp,Glu), nonpolar (e. g. Ala, VaI, Trp, Leu, He, Pro, Met, Phe, Trp) or polar (e. g. GIy, Ser, Thr, Tyr, Cys, Asn, GIn) fall within the scope of the invention as long as the activity of the StTLRP protein is not significantly, preferably not, changed, at least not changed in a negative way. In addition non-conservative amino acid substitutions fall within the scope of the invention as long as the activity of the StTLRP protein is not changed significantly, preferably not, or at least is not changed in a negative way.

Also encompassed are fragments of any of the StTLRP proteins and protein variants described above, preferably functional fragments comprising at least 10, 17, 20, 30, 40, 50, 60, 70, 80 or more consecutive amino acids of the proteins or variants. Especially the smallest active fragment which retains a biological function in the cell wall strengthening is encompassed herein. This includes hybrid and chimeric proteins comprising the smallest active fragment. Preferably, the smallest active fragment or hybrid protein comprises or consists of at least one cysteine domain or variant cysteine domain as described above. More preferably additionally at least one consensus middle domain is present.

Apart from the structural features above, the proteins can be further characterized by their biological activity in determining tissue texture characteristics when expressed in plant cells. "Tissue texture characteristics" refer to physical characteristics such as firmness, softness, graininess, crumbliness, floury and mealiness of either fresh and/or heat treated tissue (heat treatment encompasses herein cooking, steaming, microwaving, etc.). The texture characteristics can be assessed using various methods, such as touch, visual inspection, asserting pressure or tension, assessing flavour and taste, etc. The method of choice depends on the plant species in which the texture is modulated. Likewise, the suitable control tissues or organs depend on the plant species and tissue to be modified. For cooked potato tubers, texture can be rated and assigned to 6 classes, as shown in the Examples, with a score of 1 referring to the firmest tissue (non-mealy/firm) and a score of above 5 referring to the most mealy/crumbly tissue. The in vivo functionality can suitably be compared to that of SEQ ID NO: 1 and/or 2, for example by making a transgenic plant overexpressing a nucleic acid sequence according to the invention and comparing the tissue texture to transgenic plants overexpressing SEQ ID NO: 1 or 2, or to non-transgenic controls or empty vector controls.

The texture of cooked potatoes is a criterion used to classify commercial potato cultivars into "cooking types", for example A = salad potato; B = for general use; C = floury/mealy; D = very floury/mealy; nt = no typical, based for example on the following characters: desintegration, consistency, mealiness, dryness and structure according to the instruction in: "Z prac Instytutu Ziemniaka" (Instrukcja: "Ocena wartosci konsumpcyjnej ziemniakόw", 1974. Z prac Instytutu Ziemniaka, Bonin 1974). There are also intermediate evaluations, e.g. BC. A classification system for cooked potato texture is also given by the European Association of Potato Research (EAPR). Four types of cooking behaviour (A,B,C and D) are distinguished based on a sensory evaluation using the following descriptors: Mealiness, Concistency, Sloughing, Moistness and Structure (Border et al, 1986, Lebensmittel-Wissenschaft und- Technologies 9, 338-343).

Whether or not (qualitative), and to what extent (quantitative) a protein has a biological function in determining texture characteristics can be tested and compared by various methods. The most straightforward way is to transform a plant with a chimeric gene comprising the nucleic acid sequence encoding the protein, or variant or fragment, operably linked to a suitable promoter and to analyze the effect of the expression of the chimeric gene on tissue texture, compared to a suitable control tissue (e.g. a non- tranformed plant or empty-vector transformant). Generally for functional proteins according to the invention the mRNA expression levels can be correlated with increased tissue firmness. Expression levels of sttlrp genes can be analysed and quantified using known methods such as RT-PCR using sttlrp primer pairs, nucleic acid hybridization (e.g. Northern blot analysis, microarrays) and the like, or by analysing the level of StTLRP protein (using e.g. SDS-page and Western blots, ELISA assays, immunocytological assays, etc.).

Alternatively, once the allelic make-up of plants at the StTLRP locus is known (which can be determined using e.g. allele specific PCR primers or similar molecular methods as described further below), genetic methods combined with phenotypic and/or molecular methods may be used to determine whether specific alleles or allele combinations have a particular texture modulating effect. For example, a plant tissue which is homozygous for the Δ7 allele (SEQ ID NO: 1) or comprises multiple copies of this allele is going to have significantly different texture properties (firm/non-mealy) than one which is homozygous for or comprises multiple copies of the allele lacking the deletion (SEQ ID NO: 2) (mealy). Non-functional proteins will on the other hand not modulate the texture properties.

The StTLRP proteins according to the invention (including variants) may be isolated from natural sources (for example other wild accessions, breeding lines or cultivars of the species Solarium tuberosum or other species of the genus Solarium), or synthesized de novo by chemical synthesis (using e.g. a peptide synthesizer such as supplied by Applied Biosystems) or produced by recombinant host cells. The StTLRP proteins, variants or fragments according to the invention may also be used to raise mono- or polyclonal antibodies, which may for example be used for the detection of StTLRP proteins in samples (immunochemical analysis methods and kits). Preferably, they may be used to develop allele-specific detection assays.

Other putative members of the StTLRP group can be identified in silico, e.g. by identifying nucleic acid or protein sequences in existing nucleic acid or protein database (e.g. GENBANK, SWISSPROT, TrEMBL) and using standard sequence analysis software, such as sequence similarity search tools (BLASTN, BLASTP, BLASTX, TBLAST, FASTA, etc.). Especially the screening of plant sequence databases, such as the tomato, potato, rice or wheat genome databases, for the presence of amino acid sequences or nucleic acid sequences encoding the consensus domains or a sequence essentially similar to the consensus domain(s) or to the proteins of SEQ ID No:l or 2, is desired. Putative amino acid sequences or nucleic acid sequences may be selected, cloned or synthesized de novo and tested for in vivo functionality by e.g. overexpression in a plant host.

Nucleic acid sequences according to the invention

In another embodiment of the invention nucleic acid sequences (genomic DNA, cDNA and RNA) encoding the above proteins, protein variants and fragments are provided, as well as chimeric genes and vectors (expression vectors and gene silencing vectors) comprising these.

Due to the degeneracy of the genetic code various nucleic acid sequences may encode the same amino acid sequence. Any nucleic acid sequence encoding a StTLRP protein, variant or fragment thereof is referred herein to as "sttlrp". The nucleic acid sequences provided include naturally occurring, artificial or synthetic nucleic acid sequences. Examples of nucleic acid sequences encoding StTLRP are provided for in SEQ ID NO: 5-7. In addition PCR primer pairs suitable for amplifying sttltrp RNA transcripts are provided in SEQ ID NO: 8 and 9, SEQ ID NO: 10 and 11 and SEQ ID NO: 10 and 12 (specific for the Δ7 allele). Obviously, other primer pairs capable of amplifying sttlrp RNA or DNA may be designed using known methods. It is understood that when sequences are depicted as DNA sequences while RNA is referred to, the actual base sequence of the RNA molecule is identical with the difference that thymine (T ) is replace by uracil (U).

Also included are variants and fragments of sttlrp nucleic acid sequences, such as nucleic acid sequences hybridizing to sttlrp nucleic acid sequences under stringent hybridization conditions as defined. Variants of sttlrp nucleic acid sequences also include nucleic acid sequences which have a sequence identity to SEQ ID NO: 5-7 of at least 55%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, 99.8% or more. It is clear that many methods can be used to identify, synthesise or isolate variants or fragments of sttlrp nucleic acid sequences, such as nucleic acid hybridization, PCR technology, in silico analysis and nucleic acid synthesis, and the like.

The nucleic acid sequence, particularly DNA sequence, encoding the StTLRP proteins (variants or fragments) of this invention can be inserted in expression vectors to produce high amounts of StTLRP proteins (or e.g. chimeric StTLRP proteins), as described below. For optimal expression in a host the sttlrp DNA sequences can be codon-optimized by adapting the codon usage to that most preferred in plant genes, particularly to genes native to the plant genus or species of interest (Bennetzen & Hall, 1982, J. Biol. Chem. 257, 3026-3031; Itakura et al, 1977 Science 198, 1056-1063.) using available codon usage tables (e. g. more adapted towards expression in cotton, soybean corn or rice). Codon usage tables for various plant species are published for example by Ikemura (1993, In "Plant Molecular Biology Labfax", Croy, ed., Bios Scientific Publishers Ltd.) and Nakamura et al. (2000, Nucl. Acids Res. 28, 292.) and in the major DNA sequence databases (e.g. EMBL at Heidelberg, Germany). Accordingly, synthetic DNA sequences can be constructed so that the same or substantially the same proteins are produced. Several techniques for modifying the codon usage to that preferred by the host cells can be found in patent and scientific literature. The exact method of codon usage modification is not critical for this invention. Small modifications to a DNA sequence such as described above can be routinely made, i.e., by PCR-mediated mutagenesis (Ho et al., 1989, Gene 77, 51-59., White et al, 1989, Trends in Genet. 5, 185-189). More profound modifications to a DNA sequence can be routinely done by de novo DNA synthesis of a desired coding region using available techniques.

Also, the sttlrp nucleic acid sequences can be modified so that the N-terminus of the StTLRP protein has an optimum translation initiation context, by adding or deleting one or more amino acids at the N-terminal end of the protein. Often it is preferred that the proteins of the invention to be expressed in plants cells start with a Met- Asp or Met- Ala dipeptide for optimal translation initiation. An Asp or Ala codon may thus be inserted following the existing Met, or the second codon, VaI, can be replaced by a codon for Asp (GAT or GAC) or Ala (GCT, GCC, GCA or GCG). The DNA sequences may also be modified to remove illegitimate splice sites.

In another embodiment of the invention PCR primers and/or probes and kits for detecting the sttlrp DNA sequences are provided. Degenerate or specific PCR primer pairs to amplify sttlrp DNA or RNA (cDNA) from samples can be synthesized based on SEQ ID NO's 5-7, or the (reverse) complement sequences, as known in the art (see Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson at al. (2000) PCR-Basics: From Background to Bench, First Edition, Springer Verlag, Germany). Likewise, DNA fragments of SEQ ID NO's 5-7 can be used as hybridization probes. An sttlrp detection kit may comprise either sttlrp specific primers and/or sttlrp specific probes, and an associated protocol to use the primers or probe to detect sttlrp DNA and/or RNA in a sample. Such a detection kit may, for example, be used in marker assisted selection methods described below and to determine the expression levels of the alleles present in a plant at the StTLRP locus. Especially preferred are primers which allow allele-specific detection of the sttlrp alleles. For example, one or more primers which specifically detect the Δ7 allele (encoding the protein of SEQ ID NO: 1) can be designed and used to detect the mRNA expression level of this allele in plants comprising endogenous or transgenic copies of the allele and/or to transfer this allele into other plants using e.g. marker assisted selection. Various methods for allele-specific detection exist, such as for example single base extension methods (SBE; or primer extension), sequencing, fluorescent labeling assays, etc. See for example the Applied Biosystems (e.g. TaqMan Assays), SNPWave™ (a Multiplex SNP Assay) by Keygene N. V., Invader® Technology assay (Third Wave Technologies, Inc.), and many others. See also Vignal et al. (2002, Genet SeI Evol 34: 275-305).

Equally, one can determine whether a plant has been transformed with an sttlrp gene (or part thereof) of the invention. Because of the degeneracy of the genetic code, obviously amino acid codons can be replaced by others without changing the amino acid sequence of the protein.

In another embodiment antibodies that bind specifically to a StTLRP protein according to the invention are provided. In particular monoclonal or polyclonal antibodies that bind to a protein of SEQ ID NO: 1 or 2, or to fragments or variants thereof, are encompassed herein. An antibody can be prepared by using a StTLRP protein according to the invention as an antigen in an animal using methods known in the art, as e.g. described in Harlow and Lane "Using Antibodies: A laboratory manual"(New York: Cold Spring Harbor Press 1998) and in Liddell and Cryer "A Practical Guide to Monoclonal Antibodies" (Wiley and Sons, 1991). The antibodies can subsequenctly be used to isolate, identify, characterize or purify the StTLRP protein to which it binds, for example to detect the StTLRP protein in a sample, allowing the formation of an immunocomplex and detecting the presence of the immunocomplex by e.g. ELISA (enzyme linked immunoassay) or immunoblot analysis. Also provided are immunological kits, useful for detecting the StTLRP proteins, protein fragments or epitopes in a sample provided. Samples may be cells, cell supernatants, cell suspensions, tissues, etc. Such a kit comprises at least an antibody that binds to a StTLRP protein and one or more immunodetection reagents. The antibodies can also be used to isolate/identify other StTLRP proteins, for example by ELISA or Western blotting.

Chimeric genes, vectors and recombinant microorganisms according to the invention In one embodiment of the invention nucleic acid sequences encoding StTLRP proteins, as described above, are used to make chimeric genes, and vectors comprising these for transfer of the chimeric gene into a host cell and production of the StTLRP protein(s) in host cells, such as cells, tissues, organs or organisms derived from transformed cell(s). Host cells are preferably plant cells and, but microbial hosts (bacteria, yeast, fungi, etc.) are also envisaged. Any crop plant may be a suitable host, such as monocotyledonous plants or dicotyledonous plants, for example maize/corn (Zea species, e.g. Z. mays, Z. diploperennis (chapule), Zea luxurians (Guatemalan teosinte), Zea mays subsp. huehuetenangensis (San Antonio Huista teosinte), Z. mays subsp. mexicana (Mexican teosinte), Z. mays subsp. parviglumis (Balsas teosinte), Z. perennis (perennial teosinte) and Z. ramosa), wheat (Triticum species), barley (e.g. Hordeum vulgare), oat (e.g. Avena sativa), sorghum (Sorghum bicolor), rye (Secale cereale), soybean (Glycine spp, e.g. G. max), cotton (Gossypium species, e.g. G. hirsutum, G. barbadense), Brassica spp. (e.g. B. napus, B. juncea, B. oleracea, B. rapa, etc), sunflower (Helianthus annus), tobacco (Nicotiana species), alfalfa (Medicago sativa), rice (Oryza species, e.g. O. sativa indica cultivar-group or japonica cultivar-group), forage grasses, pearl millet (Pennisetum spp. e.g. P. glaucum), tree species, vegetable species, such as Lycopersicon ssp (e.g. Lycopersicon esculentum), potato (Solanum tuberosum, other Solanum species), eggplant (Solanum melongena), peppers (Capsicum annuum, Capsicum frutescens), pea, bean (e.g. Phaseolus species), fleshy fruit (grapes, peaches, plums, strawberry, mango) ornamental species (e.g. Rose, Petunia, Chrysanthemum, Lily, Gerbera species), woody trees (e.g. species of Populus, Salix, Quercus, Eucalyptus), fibre species e.g. flax (Linum usitatissimum) and hemp (Cannabis sativa).

Particularly preferred are root vegetable and tuber species and fleshy fruit producing species. Most preferred root vegetable species are sugar beet (Beta vulgaris), Brassica species (e.g. rutabaga and turnip), carrot (Daucus carota), celeriac (Apium graveolens), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), taro (Colocasia esculenta), radish (Raphanus sativus), yam (Dioscorea spp), artichoke (Helianthus tuberosus and Stachys affinis).

In one embodiment species of the genus Solanum (including the reclassified Lycopersicon) are transformed, most preferably Solanum tuberosum, such as existing cultivars or breeding lines. Especially cooking types which are mealy or slightly mealy may be transformed with an expression vector, so that a promoter active in plant cells, operably linked to a sttlrp allele, is integrated into the host's genome and expressed, preferably at high levels. Preferably the cooking type of the potato tubers is significantly modulated by expressing said allele and by increasing the mRNA levels of a nucleic acid sequence encoding a cell wall protein according to the invention. "Significantly modulated" with respect to tissue texture refers to a statistically significant change in the textural properties, especially firmness and/or mealiness. Most preferably, the change is such that the transgenic tissue falls into another class, e.g. in root vegetables or fruit (e.g. apple, tomato, etc.) a shift from mealy to firm is preferred.

The construction of chimeric genes and vectors for, preferably stable, introduction of StTLRP protein encoding nucleic acid sequences into the genome of host cells is generally known in the art. To generate a chimeric gene the nucleic acid sequence encoding a StTLRP protein is operably linked to a promoter sequence, suitable for expression in the host cells, using standard molecular biology techniques. The promoter sequence may already be present in a vector so that the sttlrp nucleic sequence is simply inserted into the vector downstream of the promoter sequence. The vector is then used to transform the host cells and the chimeric gene is inserted in the nuclear genome or into the plastid, mitochondrial or chloroplast genome and expressed there using a suitable promoter (e. g., Mc Bride et ah, 1995 Bio/Technology 13, 362; US 5,693, 507). In one embodiment a chimeric gene comprises a suitable promoter for expression in plant cells or microbial cells (e.g. bacteria), operably linked thereto a nucleic acid sequence encoding a StTLRP protein or fusion protein according to the invention, optionally followed by a 3 'nontranslated nucleic acid sequence.

The sttlrp nucleic acid sequence, preferably the sttlrp chimeric gene, encoding an functional StTLRP protein or variant, can be stably inserted in a conventional manner into the nuclear genome of a single plant cell, and the so -transformed plant cell can be used in a conventional manner to produce a transformed plant that has an altered phenotype due to the presence of the StTLRP protein in certain cells at a certain time. In this regard, a T-DNA vector, comprising a nucleic acid sequence encoding a StTLRP protein, in Agrobacterium tumefaciens can be used to transform the plant cell, and thereafter, a transformed plant can be regenerated from the transformed plant cell using the procedures described, for example, in EP 0 116 718, EP 0 270 822, PCT publication WO84/02913 and published European Patent application EP 0 242 246 and in Gould et al. (1991, Plant Physiol. 95,426-434). The construction of a T-DNA vector for Agrobacterium mediated plant transformation is well known in the art. The T-DNA vector may be either a binary vector as described in EP 0 120 561 and EP 0 120 515 or a co-integrate vector which can integrate into the Agrobacterium Ti-plasmid by homologous recombination, as described in EP 0 116 718.

Preferred T-DNA vectors each contain a promoter operably linked to StTLRP encoding nucleic acid sequence between T-DNA border sequences, or at least located to the left of the right border sequence. Border sequences are described in Gielen et al. (1984, EMBO J 3,835-845). Of course, other types of vectors can be used to transform the plant cell, using procedures such as direct gene transfer (as described, for example in EP 0 223 247), pollen mediated transformation (as described, for example in EP 0 270 356 and WO85/01856), protoplast transformation as, for example, described in US 4,684, 611, plant RNA virus- mediated transformation (as described, for example in EP 0 067 553 and US 4,407, 956), liposome-mediated transformation (as described, for example in US 4,536, 475), and other methods such as those described methods for transforming certain lines of corn (e. g., US 6,140, 553; Fromm et al., 1990, Bio/Technology 8, 833-839; Gordon-Kamm et al., 1990, The Plant Cell 2, 603-618) and rice (Shimamoto et al., 1989, Nature 338, 274-276; Datta et al. 1990, Bio/Technology 8, 736-740) and the method for transforming monocots generally (PCT publication WO92/09696). For cotton transformation see also WO 00/71733, and for rice transformation see also the methods described in W092/09696, W094/00977 and W095/06722. For sorghum transformation see e.g. Jeoung JM et al. 2002, Hereditas 137: 20-8 or Zhao ZY et al. 2000, Plant MoI Biol.44:789-98). Likewise, selection and regeneration of transformed plants from transformed cells is well known in the art. Obviously, for different species and even for different varieties or cultivars of a single species, protocols are specifically adapted for regenerating transformants at high frequency.

Besides transformation of the nuclear genome, also transformation of the plastid genome, preferably chloroplast genome, is included in the invention. One advantage of plastid genome transformation is that the risk of spread of the transgene(s) can be reduced. Plastid genome transformation can be carried out as known in the art, see e.g. Sidorov VA et al 1999, Plant J.19: 209-216 or Lutz KA et al 2004, Plant J. 37(6):906- 13.

The resulting transformed plant can be used in a conventional plant breeding scheme to produce more transformed plants with the same characteristics or to introduce the gene part into other varieties of the same or related plant species. Seeds, which are obtained from the transformed plants, contain the chimeric sttlrp gene as a stable genomic insert. Cells of the transformed plant can be cultured in a conventional manner to produce the StTLRP protein, which can be recovered for other use e.g. antibody production.

The sttlrp nucleic acid sequence is inserted in a plant cell genome so that the inserted coding sequence is downstream (i.e. 3') of, and under the control of, a promoter which can direct the expression in the plant cell. This is preferably accomplished by inserting the chimeric gene in the plant cell genome, particularly in the nuclear or plastid (e. g. chloroplast) genome.

Preferred promoters include: the strong constitutive 35S promoters or enhanced 35S promoters (the "35S promoters") of the cauliflower mosaic virus (CaMV) of isolates CM 1841 (Gardner et al., 1981, Nucleic Acids Research 9, 2871-2887), CabbB-S (Franck et al., 1980, Cell 21, 285-294) and CabbB-JI (Hull and Howell, 1987, Virology 86,482-493); the 35S promoter described by Odell et al. (1985, Nature 313, 810-812) or in US5164316, promoters from the ubiquitin family (e.g. the maize ubiquitin promoter of Christensen et al., 1992, Plant MoI. Biol. 18,675-689, EP 0 342 926, see also Cornejo et al. 1993, Plant Mol.Biol. 23, 567-581), the gos2 promoter (de Pater et al, 1992 Plant J. 2, 834-844), the emu promoter (Last et al, 1990, Theor. Appl. Genet. 81,581-588), Arabidopsis actin promoters such as the promoter described by An et al (1996, Plant J. 10, 107.), rice actin promoters such as the promoter described by Zhang et α/.(1991, The Plant Cell 3, 1155-1165) and the promoter described in US 5,641,876 or the rice actin 2 promoter as described in WO070067; promoters of the Cassava vein mosaic virus (WO 97/48819, Verdaguer et al 1998, Plant MoI. Biol. 37,1055-1067), the pPLEX series of promoters from Subterranean Clover Stunt Virus (WO 96/06932, particularly the S7 promoter), a alcohol dehydrogenase promoter, e.g., pAdhlS (GenBank accession numbers X04049, X00581), and the TRl' promoter and the TR2' promoter (the "TRl'promoter" and "TR2'promoter", respectively) which drive the expression of the 1' and 2' genes, respectively, of the T-DNA (Velten et al., 1984, EMBO J 3, 2723-2730), the Figwort Mosaic Virus promoter described in US6051753 and in EP426641, histone gene promoters, such as the Ph4a748 promoter from Arabidopsis (PMB 8: 179-191), or others.

Alternatively, a promoter can be utilized which is not constitutive but rather is specific for one or more tissues or organs of the plant (tissue preferred / tissue specific, including developmentally regulated promoters), for example leaf preferred, epidermis preferred, root preferred, flower tissue e.g. tapetum or anther preferred, seed preferred, pod preferred, etc.), whereby the sttlrp gene is expressed only in cells of the specific tissue(s) or organ(s) and/or only during a certain developmental stage. For example, the sttlrp gene(s) can be selectively expressed in the leaves of a plant by placing the coding sequence under the control of a light-inducible promoter such as the promoter of the ribulose-1, 5-bisphosphate carboxylase small subunit gene of the plant itself or of another plant, such as pea, as disclosed in US 5,254, 799 or Arabidopsis as disclosed in US5034322.

The choice of the promoter is determined by the phenotype one aims to achieve, as will be described in more detail below. For example, to achieve fruits (e.g. tomatoes) with modulated texture properties (especially increased firmness and/or less mealiness) and therefore a firmer / less mealy fruit flesh, a fruit specific or fruit preferred promoter is the most suitable. A tomato fruit and peel specific promoter is for example the promoter of. beta-Galactosidase II (Smith et al, 1998, Plant Physiol 117: 417-23). Other promoters include the promoter from fruit specific invertase genes or polygalacturonase genes.

For certain phenotypes such as potatoes (i.e. tubers) with modulated texture properties, especially increased firmness / decreased mealiness a tuber specific promoter is most suitable, for example, the GBSS promoter (visser et al 1991, Plant MoLBiol. 17: 691- 699) or the patatin promoter (Nap et al 1992, Plant MoLBiol. 20: 683-694). For modulating texture in root tissue, a promoter preferentially active in roots is described in WO00/29566. Another promoter for root preferential expression is the ZRP promoter (and modifications thereof) as described in US 5,633, 363.

For modulating expression in any of the root vegetables (as defined) a promoter active (at least) in the particular underground storage tissue of the plant can be used or identified.

Another alternative is to use a promoter whose expression is inducible. Examples of inducible promoters are wound- inducible promoters, such as the MPI promoter described by Cordera et al. (1994, The Plant Journal 6, 141), which is induced by wounding (such as caused by insect or physical wounding), or the COMPTII promoter (WO0056897) or the promoter described in US6031151. Alternatively the promoter may be inducible by a chemical, such as dexamethasone as described by Aoyama and Chua (1997, Plant Journal 11: 605-612) and in US6063985 or by tetracycline (TOPFREE or TOP 10 promoter, see Gatz, 1997, Annu Rev Plant Physiol Plant MoI Biol. 48: 89-108 and Love et al. 2000, Plant J. 21: 579-88) or ethanol (see the ethanol- inducible promoter system described in Ait-ali et al., 2001, Plant Biotechnology Journal 1, 337-343, wherein ethanol treatment activates alcR, which in turn induces expression of the alc:35S promoter). Other inducible promoters are for example inducible by a change in temperature, such as the heat shock promoter described in US 5,447, 858, by anaerobic conditions (e.g. the maize ADHlS promoter), by light (US6455760), by pathogens (e.g. EP759085 or EP309862) or by senescence (SAG12 and SAG13, see US5689042). Obviously, there are a range of other promoters available. A podwall specific promoter from Arabidopsis is the FUL promoter (also referred to as AGL8 promoter, WO9900502; WO9900503; Liljegren et al. 2004 Cell.116(6):843-53)), the Arabidopsis INDl promoter (Lijegren et al. 2004, supra.; WO9900502; WO9900503) or the dehiscence zone specific promoter of a Brassica polygalacturonase gene (WO9713856). A petal specific promoter has been described in WO9915679. Seed specific promoters are described in EP723019, EP255378 or WO9845461. Specific expression of an StTLRP allele in oilseed rape (Brassica napus), in the dehiscence zone, may for example enhance podshatter ressitance, and is an embodiment of the invention. The StTLRP coding sequence is inserted into the plant genome so that the coding sequence is upstream (i.e. 5') of suitable 3'end transcription regulation signals ("3 'end")

(i.e. transcript formation and polyadenylation signals). Polyadenylation and transcript formation signals include those of the CaMV 35S gene ("3' 35S"), the nopaline synthase gene ("3' nos") (Depicker et al, 1982 J. Molec. Appl. Genetics 1, 561-573.), the octopine synthase gene ("3'ocs") (Gielen et al, 1984, EMBO J 3, 835-845) and the

T-DNA gene 7 ("3' gene 7") (Velten and Schell, 1985, Nucleic Acids Research 13,

6981-6998), which act as 3 '-untranslated DNA sequences in transformed plant cells, and others.

Introduction of the T-DNA vector into Agrobacterium can be carried out using known methods, such as electroporation or triparental mating.

A StTLRP encoding nucleic acid sequence can optionally be inserted in the plant genome as a hybrid gene sequence whereby the sttlrp sequence is linked in-frame to a (US 5,254, 799; Vaeck et al, 1987, Nature 328, 33-37) gene encoding a selectable or scorable marker, such as for example the neo (or nptll) gene (EP 0 242 236) encoding kanamycin resistance, so that the plant expresses a fusion protein which is easily detectable.

When reference to a transgenic plant cell is made herein, this refers to a plant cell (or also a plant protoplast) as such in isolation or in tissue culture, or to a plant cell (or protoplast) contained in a plant or in a differentiated organ or tissue, and both possibilities are specifically included herein. Hence, a reference to a plant cell in the description or claims is not meant to refer only to isolated cells in culture, but refers to any plant cell, wherever it may be located or in whatever type of plant tissue or organ it may be present.

All or part a sttlrp nucleic acid sequence, encoding a StTLRP protein, can also be used to transform microorganisms, such as bacteria (e.g. Escherichia coli, Pseudomonas, Agrobacterium, Bacillus, etc.), fungi, viruses, algae or insects. Transformation of bacteria, with all or part of a sttlrp nucleic acid sequence of this invention, incorporated in a suitable cloning vehicle, can be carried out in a conventional manner, preferably using conventional electroporation techniques as described in Maillon et al. (1989, FEMS Microbiol. Letters 60, 205-210.) and WO 90/06999. For expression in prokaryotic host cell, the codon usage of the nucleic acid sequence may be optimized accordingly (as described for plants above). Intron sequences should be removed and other adaptations for optimal expression may be made as known.

For obtaining enhanced expression in monocot plants such as grass species, e.g. corn or rice, an intron, preferably a monocot intron, can be added to the chimeric gene. For example the insertion of the intron of the maize Adhl gene into the 5' regulatory region has been shown to enhance expression in maize (Callis et. al., 1987, Genes Develop. 1: 1183-1200). Likewise, the HSP70 intron, as described in US 5,859, 347, may be used to enhance expression. The DNA sequence of the sttlrp nucleic acid sequence can be further changed in a translationally neutral manner, to modify possibly inhibiting DNA sequences present in the gene part by means of site-directed intron insertion and/or by introducing changes to the codon usage, e. g., adapting the codon usage to that most preferred by plants, preferably the specific relevant plant genus, as described above.

In accordance with one embodiment of this invention, the StTLRP proteins (or variant, fragment or chimeric proteins) are targeted to intracellular organelles such as plastids, preferably chloroplasts, mitochondria, or vacuoles. More preferably the proteins are targeted to the cell wall / extracellular space via a secretion signal peptide. To allow secretion of the StTLRP proteins to the outside of the transformed host cell, an appropriate secretion signal peptide may be fused to the amino terminal end (N- terminal end) of the StTLRP protein or replace the native putative secretion peptide. Putative signal peptides can be detected using computer based analysis, using programs such as the program Signal Peptide search (SignalP Vl.1 or 2.0)(Von Heijne, Gunnar, 1986 and Nielsen et al, 1996).

For this purpose, in one embodiment of this invention, the chimeric genes of the invention comprise a coding region encoding a signal or target peptide, linked to the StTLRP protein coding region of the invention. Preferably, the peptide is the natural signal peptide already present in the proteins or a peptide from a variant or ortholog. Alternatively, a heterologous secretion signal peptide may be added or used to replace the native one. For example the secretion signal of the potato proteinase inhibitor 1 (Keil et al., 1986, Nucl. Acids Res. 14,5641-5650), the secretion signal of the alpha- amylase 3 gene of rice (Sutliff et al, 1991, Plant Molec. Biol. 16,579-591) and the secretion signal of tobacco PRl protein (Cornelissen et al, 1986, EMBO J. 5,37-40) may be used.

Signal sequences for targeting to intracellular organelles or for secretion outside the plant cell or to the cell wall are found in naturally targeted or secreted proteins, preferably those described by Klosgen et al. (1989, MoI. Gen. Genet. 217, 155-161), Klosgen and Weil (1991, MoI. Gen. Genet. 225, 297-304), Neuhaus & Rogers (1998, Plant MoI. Biol. 38, 127-144), Bih et al. (1999, J. Biol. Chem. 274, 22884-22894), Morris et al. (1999, Biochem. Biophys. Res. Commun. 255, 328-333), Hesse et al. (1989, EMBO J. 8, 2453-2461), Tavladoraki et al. (1998, FEBS Lett. 426,62-66.), Terashima et al. (1999, Appl. Microbiol. Biotechnol. 52,516-523), Park et al. (1997, J.Biol. Chem. 272, 6876-6881), Shcherban et al. (1995, Proc. Natl. Acad. Sci USA 92,9245-9249).

In one embodiment, several StTLRP encoding nucleic acid sequences are co-expressed in a single host. A co-expressing host plant is easily obtained by transforming a plant already expressing StTLRP protein of this invention, or by crossing plants transformed with different StTLRP proteins of this invention. Alternatively, several StTLRP protein encoding nucleic acid sequences can be present on a single transformation vector or be co -transformed at the same time using separate vectors and selecting transformants comprising both chimeric genes. Similarly, one or more StTLRP encoding genes may be expressed in a single plant together with other chimeric genes, for example encoding other proteins involved in mechanical strength of cell walls and tissue texture, such as for example pectin methyl esterases, xylosidases, endoglucanases, expansins, etc.

It is understood that the different proteins can be expressed in the same plant, or each can be expressed in a single plant and then combined in the same plant by crossing the single plants with one another. For example, in hybrid seed production, each parent plant can express a single protein. Upon crossing the parent plants to produce hybrids, both proteins are combined in the hybrid plant.

Preferably, for selection purposes but also for weed control options, the transgenic plants of the invention are also transformed with a DNA encoding a protein conferring resistance to herbicide, such as a broad-spectrum herbicide, for example herbicides based on glufosinate ammonium as active ingredient (e.g. Liberty® or BASTA; resistance is conferred by the PAT or bar gene; see EP 0 242 236 and EP 0 242 246) or glyphosate (e.g. RoundUp®; resistance is conferred by EPSPS genes, see e.g. EPO 508 909 and EP 0 507 698). Using herbicide resistance genes (or other genes conferring a desired phenotype) as selectable marker further has the advantage that the introduction of antibiotic resistance genes can be avoided.

Alternatively, other selectable marker genes may be used, such as antibiotic resistance genes. As it is generally not accepted to retain antibiotic resistance genes in the transformed host plants, these genes can be removed again following selection of the transformants. Different technologies exist for removal of transgenes. One method to achieve removal is by flanking the chimeric gene with lox sites and, following selection, crossing the transformed plant with a CRE recombinase-expressing plant (see e.g. EP506763B1). Site specific recombination results in excision of the marker gene. Another site specific recombination systems is the FLP/FRT system described in EP686191 and US5527695. Site specific recombination systems such as CRE/LOX and FLP/FRT may also be used for gene stacking purposes. Further, one-component excision systems have been described, see e.g. WO9737012 or WO9500555).

Gene- silencing / downregulation and loss-of-function

It is also an embodiment of the invention to downregulate endogenous sttlrp genes or gene families in plants or plant parts or to express a dominant loss-of-function fusion protein, for example in order to reduce tissue firmness and/or enhance softness and/or mealiness of tissues and organs.

Downregulation can be achieved by using gene silencing approaches, to which essentially the same methods apply as above, with the exception that instead of expression vectors gene-silencing vectors are used. "Gene silencing" refers to the down-regulation or complete inhibition of gene expression of one or more sttlrp target genes. The use of inhibitory RNA to reduce or abolish gene expression is well established in the art and is the subject of several reviews (e.g Baulcombe 1996, Stam et al. 1997, Depicker and Van Montagu, 1997). There are a number of technologies available to achieve gene silencing in plants, such as chimeric genes which produce antisense RNA of all or part of the target gene (see e.g. EP 0140308 Bl, EP 0240208 Bl and EP 0223399 Bl), or which produce sense RNA (also referred to as co- suppression), see EP 0465572 Bl.

The most successful approach so far has however been the production of both sense and antisense RNA of the target gene ("inverted repeats"), which forms double stranded RNA (dsRNA) in the cell and silences the target gene. Methods and vectors for dsRNA production and gene silencing have been described in EP 1068311, EP 983370 Al, EP 1042462 Al, EP 1071762 Al and EP 1080208 Al.

A vector according to the invention may therefore comprise a transcription regulatory region which is active in plant cells operably linked to a sense and/or antisense DNA fragment of a sttlrp gene according to the invention. Generally short (sense and antisense) stretches of the target gene sequence, such as 17, 18, 19, 20, 21, 22 or 23 nucleotides of cording or non-coding sequence are sufficient. Longer sequences can also be used, such as 100, 200 or 250 nucleotides. Preferably, the short sense and antisense fragments are separated by a spacer sequence, such as an intron, which forms a loop (or hairpin) upon dsRNA formation. Any short stretch of SEQ ID NO: 5-7 or variants thereof may be used to make an sttlrp gene silencing vector and a transgenic plant in which one or more sttlrp genes are silenced in all or some tissues or organs. A convenient way of generating hairpin constructs is to use generic vectors such as pHANNIBAL and pHELLSGATE, vectors based on the Gateway® technology (see Wesley et al 2004, Methods MoI Biol. 265:117-30; Wesley et al. 2003, Methods MoI Biol. 236:273-86 and Helliwell & Waterhouse 2003, Methods 30(4):289-95.), all incorporated herein by reference.

By choosing conserved nucleic acid sequences all sttlrp group members, the entire gene group in a host plant can be silenced. Encompassed herein are also transgenic plants comprising a transcription regulatory element operably linked to a sense and/or antisense DNA fragment of a sttlrp gene and exhibiting a sttlrp gene silencing phenotype.

In one embodiment sttlrp gene silencing is used to generate host plants comprising less firm (softer) and/or more mealy tissue, especially root vegetables, compared to suitable controls. However, due to structural and functional redundancy, gene silencing approaches may not always be successful and may show no phenotypic change or only a subtle phenotype, possibly revealed only under extreme environmental conditions, when knocked-out. A different approach is, therefore, to generate plants having modulated tissue texture characteristics by over-expressing a StTLRP -repressor domain fusion protein in the host cells. In a preferred embodiment this chimeric protein is a StTLRP-EAR fusion protein or a En-StTLRP fusion protein, e.g. a En²⁹⁸-StTLRP fusion protein.

For example, StTLRP protein fusions are made with a 12 amino acid 'EAR' repressor domain as described by Hiratsu et ah, 2003 (Plant J. 34:733-739), incorporated herein by reference. These repressor domain fusions to any one of the StTLRP proteins (as defined), termed herein 'StTLRP-EAR' fusion proteins, are able to cause repression of the downstream target genes and thus result in an effective loss-of-function mutant (dominant negative effect). To generate a StTLRP -repressor domain fusion protein, the nucleic acid sequence encoding the repressor domain is translationally fused to the nucleic acid sequence comprising the StTLRP coding sequence. The StTLRP -repressor domain fusion protein encoding nucleic acid sequence (especially StTLRP-EAR) is placed under control of constitutive or specific promoters (e.g. tissue specific or developmentally regulated).

It is understood that StTLRP proteins may be operably fused to other repression domain available in the art which function in plant cells. These include repressor domains of animal proteins, such as the Drosophila ENGRAILED (En) repressor domain. For example the N-terminal 298 amino acids may be fused to a StTLRP protein according to the invention, creating a dominant-negative chimeric protein (see Markel et al. 2002, Nucleic Acid Research VoI 30, 4709-4719 and Chandler and Werr 2003, Trends in Plant Science Vol. 8, 279-285, both incorporated by reference). It is noted that repressor domains may be fused to the StTLRP protein at the C-terminus or at the N-terminus, depending on the domain. The nucleic acid sequence encoding the dominant-negative fusion protein may be referred to as a "dominant-negative chimeric gene" and when transferred into a host genome as a "dominant-negative transgene" (either stably integrated in the host genome or transiently expressed). Other plant repressor domains are for example the LEUNG and SEUSS co-repressors of AGAMOUS, FLC and poly comb proteins. Other animal repressor domains include for example the WTl, eve, c-ErbA and v-ErbA and Kruppel associated box (see Chandler and Werr, 2003, supra and references therein).

Transformed plant cells/plants/seeds and uses of the nucleic acid sequence and proteins according to the invention

In the following part the use of the sttlrp sequences according to the invention to generate transgenic plant cells, plants, plant seeds and any derivatives/progeny thereof, with modulated texture characteristics are described. Although any plant may be transformed, the most preferred examples are described.

In general, a transgenic plant or plant tissue or organ according to the invention, and having modulated texture characteristics compared to suitable controls, comprises, preferably integrated in its genome, a chimeric gene comprising a transcription regulatory sequence active in plant cells, operably linked to a nucleic acid sequence selected from the group of:

(a) a nucleic acid sequence encoding a protein of SEQ ID NO: 1 or 2; (b) a nucleic acid sequence encoding a protein having at least 77%, preferably at least

80%, or more, amino acid identity to SEQ ID NO: 1 and/or 2 over the entire length; or at least 70%, preferably at least 77% or more amino acid sequence identity over the entire length to SEQ ID NO: 3 and/or 4;

(c) a sense and/or antisense fragment of the nucleic acid sequence of (a) or (b), or the complementary sequences thereof.

A) Potato plants with modulated texture properties A transgenic potato plant, whose tubers have modulated texture properties can be made as described above, by transforming a potato host cell with an sttlrp nucleic acid sequence, operably linked to a promoter active in plant cells.

The transgenic potato plant may be of any ploidy, such as diploid (2n = 24), hexaploid, triploid, tetraploid or pentaploid. As the cultivated potato is autotetraploid (4n=48), it is preferred to transform autotetraploid clones or varieties.

In one embodiment transgenic plants are provided, comprising within their genome a chimeric gene which comprises a promoter (e.g. a tuber specific promoter) operably linked to a StTLRP protein encoding DNA sequence according to the invention. Also provided are the mature tubers of those plants, as well as seed potatoes and progeny of any of these. Both fresh, harvested tubers as well as processed tubers are provided, such as cooked or partially cooked, cut (sliced, diced, etc.) and/or packaged.

The texture of these tubers is preferably firm after heat treatment (such as steaming), and preferably comprises a visual score of 3 or less, more preferably 2 or less, most preferably 1 (on a scale of 1-6 as described in the Examples, with 1 being firm/non- mealy). Texture firmness is preferably increased relative to the control by at least 1%, 2%, 5%, more preferably at least 10% or more. In addition, mRNA expression of one or more sttlrp alleles is preferably increased by at least 3-fold, 5-fold, 10-fold, 20-fold 40-fold, 50-fold 60-fold or more relative to the non-transgenic controls.

Preferably one or more high expressing sttlrp alleles are used to transform potato plants. Especially preferred are alleles comprising one or more (e.g. 7 or 8) amino acid deletions in the cysteine domain (as described; e.g. the cysteine domain of SEQ ID NO:

I)-

In another embodiment, gene silencing or dominant negative approaches are used to enhance mealines and/or reduce firmness (enhance softness) of root vegetables, such as potato tubers. Texture firmness of the fresh and/or heat treated tissue is preferably reduced relative to the control by at least 1%, 2%, 5%, more preferably at least 10% or more. B) Other root vegetables having modulated texture properties

As for potato tubers above, it is also an embodiment of the invention to provide other root vegetables having modified tissue texture characteristics. Transgenic plants which overexpress a functional StTLRP protein or a StTLRP -repressor domain fusion protein can be made as described. Similarly, plants transformed with an sttlrp gene silencing construct can be made as described.

The tissue texture of at least the harvestable plant parts is modulated to be either significantly firmer / less mealy (due to sttlrp overexpression) or significantly softer / more mealy due to sttlrp silencing or overexpression of StTLRP -repressor domain fusion proteins. Thus, for example sugar beet roots, Cassava, yam, sweet potato, turnips, etc. having modulated texture characteristics are provided. Tissue texture firmness of the fresh and/or heat treated tissue is preferably reduced or increased relative to the control by at least 1%, 2%, 5%, more preferably at least 10% or more.

Both firmer and softer root vegetables have particular uses, for example on the fresh market, but also in the food processing industry. In addition, firmer root vegetables can be transported without significant tissue damage and stored longer.

C) Fruit comprising modified texture characteristics

In another embodiment the texture of the transgenic fruit is modified compared to the fruit of non- transgenic plants. The firmness of the fruit flesh is preferably significantly enhanced and/or mealiness is significantly reduced (compared to controls) by expressing one or more sttlrp alleles in at least the fruit tissue. As in the examples above, mRNA levels of sttlrp transcripts are preferably significantly increased.

The texture characteristics can be determined in either fresh fruit tissue or in heat treated tissue, depending on the species. For example tomato fruit may be evaluated fresh. Firmness may be evaluated using any means. Mealiness is preferably assessed using sensory assays, whereby sensory panels are asked to score taste characteristics.

Any fleshy fruit may comprise the transgenes according to the invention. Preferred fruit are tomato, apple, plums, bananas, peach, nectarine, apricot, mango, pear, melon, parsimon, etc. Especially preferred are processing and cooking types (such as cooking apples, processing tomatoes).

The invention comprises both fresh (harvested and non-harvested) and processed fruit, such as (partially) cooked, sliced, diced, juice, paste, etc.

In a preferred embodiment the host plant is a tomato plant {Lycopersicon species) and the modified fruit is a tomato. Lycopersicon species include L. cheesmanii, L. chilense, L. chmielewskii, L. esculentum (tomato), Lycopersicon esculentum var. cerasiforme (cherry tomato), L. esculentum x L. peruvianum, L. glandulosum, L. hirsutum, L. minutum, L. parviflorum, Lycopersicon pennellii, L. peruvianum (Peruvian tomato), L. peruvianum var. humifusum and L. pimpinellifolium (currant tomato).

The transgenic fruit will be firmer, less mealy and fruit will also be easier and cost effective to transport with less damage and spoilage. In addition cold storage time can be increased.

Alternatively, any plant host producing fleshy fruit, for example grape, peach, plum, cherry, mango, strawberry can be transformed in order to modify the flesh texture and reduced post-harvest damage.

Suitable fruit specific promoters or promoters specifically expressed during fruit development and/or in a certain cells/tissues of the fruit are known in the art. Other suitable promoters can be easily identified by a person skilled in the art. For example, for each fleshy fruit, a promoter active in the fleshy tissue can be identified.

In the same way as already described under A and B above, it is also envisaged to make transgenic fruit which are softer and/or mealier than non-transgenic controls. Such softer fruit may be especially suitable for particular processing methods, such as preparation of fruit juices, etc.

Whole plants, seeds, cells, tissues and progeny (such as Fl, F2 seeds/plants, etc) of any of the transformed plants described above are encompassed herein and can be identified by the presence of the transgene in the DNA, for example by PCR analysis. Also "event specific" PCR diagnostic methods can be developed, where the PCR primers are based on the plant DNA flanking the inserted chimeric gene, see US6563026. Similarly, event specific AFLP fingerprints or RFLP fingerprints may be developed which identify the transgenic plant or any plant, seed, tissue or cells derived there from.

It is understood that the transgenic plants according to the invention preferably do not show non-desired phenotypes, such as yield reduction, enhanced susceptibility to diseases or undesired architectural changes (dwarfing, deformations) etc. and that, if such phenotypes are seen in the primary transformants, these can be removed by normal breeding and selection methods (crossing / backcrossing / selfing, etc.). Any of the transgenic plants described herein may be homozygous or hemizygous for the transgene.

Non-transgenic plants comprising a modified texture characteristics and methods for making these

In yet a different embodiment, non transgenic plants having modified texture characteristics are provided, as well as methods for making these.

As it was found that an increase in sttlrp expression levels correlates with an increase in tissue firmness and/or a reduction in mealiness, the methods are based on the identification and/or selection of (preferably high expressing) sttlrp alleles and the use of molecular assays for transferring these to other plants and/or for combining particular alleles in plants.

Therefore, in its broadest sense, a method for modulating texture characteristics of plant tissues or plant organs is provided, wherein the method comprises: increasing and/or decreasing the mRNA expression levels of a nucleic acid sequence encoding a cell wall protein in said plant tissues or organs, characterized in that said cell wall protein comprises at least 80% amino acid sequence identity over the entire length to SEQ ID NO: 1 or SEQ ID NO: 2. The mRNA expression level can be measured by using e.g. the PCR primers provided herein (e.g. SEQ ID NO: 8 and 9) or other primers designed based on sttlrp nucleic acid sequences. To assess the mRNA levels it is not necessary to distinguish between different alleles and the total sttlrp mRNA level may be assessed in the tissue to be modified (such as the tuber or fruit flesh). Alternatively, allele specific methods may be used to assess the expression of a particular allele.

The mRNA expression levels are modulated by marker assisted selection of one or more alleles of a nucleic acid sequence encoding a cell wall protein comprising at least 80% amino acid sequence identity over the entire length to SEQ ID NO: 1 or SEQ ID NO: 2.

"Modulated" with respect to mRNA expression levels refers herein to a significant increase and/or decrease of either the total sttlrp mRNA levels in the tissue and/or of the sttlrp mRNA of one or more specific alleles, such as those encoding SEQ ID NO: 1 and/or 2. "Significant" refers to an increase and/or decrease of mRNA levels of at least 3-fold, 5-fold, preferably at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold or more compared to one or both of the parent plants used or to control plants having a known tissue firmness (such as for potato the firm variety Nicola and the mealy variety Irene). Preferably, the same tissue type and age are used for comparisons.

The increased / decreased expression of one or more alleles (or of the total sttlrp mRNA) should be sufficient to alter the textural characteristics of the plants, i.e. making the tissue firmer and/or less mealy or making the tissue softer and/or more mealy.

To increase and/or decrease mRNA expression levels by marker assisted selection one carries out the following steps:

(a) identify (a plant comprising) one or more desired alleles of ^'sttlrp nucleic acid sequence, preferably alleles which have a high mRNA expression level or which encode functional StTLRP proteins (or variants) capable of conferring the desired tissue texture characteristics;

(b) develop molecular markers (and a molecular assay) specific for said allele(s); (c) use said molecular markers (and said assay) to transfer and/or combine one or more (preferably high expressing) alleles to/in progeny of a cross between at least one parent plant comprising said desired (high expressing) allele and another plant; and optionally

(d) testing the mRNA expression level and/or the texture characteristics of the progeny of said cross at one or more times; and optionally

(e) identifying progeny comprising said (preferably high expressing) alleles and/or allele combinations and/or modulated texture characteristics.

Optionally progeny having an optimal allele combination may be used further in breeding schemes or in the above method.

Preferably (plants comprising) high expressing alleles such as the one encoding the protein of SEQ ID NO: 1 or variants thereof, are identified in a plant in step (a). This can be done by using the sequences provided herein, for example by PCR amplification, nucleic acid hybridization, etc. For example, the Solarium tuberosum plant described in the Examples as parent E comprises such an allele. All kinds of plants can be screened for sttlrp expression levels and their allelic make-up with respect to the sttlrp genes. For example natural populations, wild accessions, varieties, breeding lines and cultivars can be analyzed and the nucleic acid and protein sequence of the sttlrp genes determined. Apart from natural alleles, one can also mutagenize plant tissue (e.g. seeds) to generate mutant alleles (see further below).

In step (b) molecular markers and a molecular assay is developed or identified which can be used to select for the presence or absence of the allele in plant tissue. The markers may be any kind of molecular markers, such as RFLP, CAPS (cleaved amplified polymorphic sequence assay, see Akopyanz et al, Nucleic Acid Research, 20:6221-6225, 1992; and Konieczny & Ausubel, The Plant Journal, 4:403-410, 1993), AFLP, SNP, any PCR based markers, probes, etc. as long as one or more alleles at the locus can be selected for using said markers. For example the sequence of the gene / transcript itself may be used as marker. Thus, it is clear that any reference to "molecular marker" herein encompasses the genomic, cDNA and RNA of the sttlrp gene itself. The molecular marker is preferably "allele specific", meaning that one can discriminate between different alleles using the marker. Allele specific markers generally detect polymorphisms between alleles (e.g. single nucleotide polymorphisms or deletions, etc.). To develop an allele discriminating assay one therefore preferably compares the nucleic acid sequences of sttlrp alleles. Two allelic variants of the potato sttlrp gene are already provided herein (SEQ ID NO: 5 and 6, encoding the proteins of SEQ ID NO: 1 and 2, respectively), but the skilled person can easily identify and sequence other alleles from potato or orthologs from other species and develop markers based on these sequences.

In one embodiment the molecular marker assay is capable of discriminating between the presence in the genome of the nucleic acid sequences encoding the protein of SEQ

ID NO: 1 and other alleles, such as the nucleic acid sequence encoding a protein comprising a full length cysteine domain (e.g. SEQ ID NO: 2). For example, PCR primers may be designed in such a way that they only create an amplification product

(using a genomic or cDNA template) when the nucleic acid sequence encoding SEQ ID NO: 1 is present or is expressed. An example of such a primer pair is provided herein in

SEQ ID NO: 10 and 12. In this way the absence or presence of this allele in the genome can be determined.

It is especially desired to discriminate high expressing alleles from other, lower expressing alleles. Without limiting the invention it is thought that high expressing allele comprising one or more amino acid deletions in the cysteine domain having the sequence C(X)_2-3 (C)_1-2 (X)_4-6 Co_-1(X)_0-3CC, wherein X is any amino acid and C is cysteine. However, it is also possible that high expressing alleles are expressed at a higher level because they comprise different transcription regulatory elements (e.g. the promoter may be more active or repressors may be absent) compared to lower expressing alleles.

Alternatively, markers may be linked in cis to the gene, i.e. they may be further upstream and/or downstream, flanking the locus, e.g. between AFLP markers E32/M49 and E32/M48 or E32/M54 on chromosome 9 of potato (see Examples). Thus, certain polymorphisms (e.g. SNPs) in these flanking regions may be associated (linked) to a specific sttlrp allele and can be used to devise a molecular assay for this allele. However, the risk of using more distant markers is that recombination events occur between the gene and the marker, so that selection of the marker is not effective anymore.

In step (c) the plant is then crossed with another plant of the same species (or a more distantly related species, as long as progeny can be recovered). The other plant preferably (although not necessarily) lacks this allele. For example, a potato plant which produces mealy tubers likely lacks the allele and would therefore benefit from its introduction.

The progeny of the cross are then analyzed for the presence and absence of the marker (using the molecular marker assay) and plants are selected and identified which comprise the desired allele(s). The progeny analyzed may be Fl hybrids, F2 or F3 families, etc. and/or backcross populations, as desired. Also, obviously marker assisted selection may be repeated several times, e.g. in different generations of the breeding scheme. In addition or instead, marker assisted selection may also be used to select against certain alleles, e.g. low expressing alleles, in order to remove these from a breeding line.

Independent of the molecular assay used, it will generally involve the taking tissue samples of the progeny plants. These may then be used to extract the genomic DNA or macerated and used directly in a PCR reaction. The exact molecular method is not relevant herein, as any assay format may be used.

Step (d), which is optional, involves testing, and optionally quantifying, the mRNA expression level of the sttlrp allele(s) and/or the texture characteristics of the progeny of said cross at one or more times during the procedure. Based hereon further selections of individual plants can be made, while others are discarded.

Step (e) is also optional and involves identifying progeny comprising the (preferably high expressing) alleles and/or allele combinations and/or modulated texture characteristics at one or more times. The aim is, obviously, to identify progeny plants having the desired allelic make-up and texture properties. Using molecular markers selection can be speeded up significantly, as phenotypic assays (e.g. texture assays) are reduced or essentially eliminated.

The identified progeny may, thus, for example comprise the following allelic make up:

- homozygous for a high expressing allele, e.g. Δ7/ Δ7 (encoding SEQ ID NO: 1 / SEQ ID NO: 1) or other combinations;

- heterozygous for a high expressing allele and a lower expressing allele, e.g. Δ7 (SEQ ID NO: 1) /SEQ ID NO: 2; - homozygous or heterozygous for alleles which encode identical or essentially similar StTLRP proteins, but wherein the endogenous transcription regulatory elements differ and result in higher expression of at least one allele.

Alternatively, the sttlrp alleles do not necessarily differ significantly in expression, but they encode StTLRP proteins or protein variants which have improved in vivo functionality, for example they are capable of conferring stronger cell walls and firmer tissue compared to e.g. the protein of SEQ ID NO: 2.

Therefore, breeding schemes can be reduced in length by several years. Descriptions of breeding methods that are commonly used for different traits and crops, as well as specifically for tomato, can be found in one of several reference books (i.e., Allard,

R. W., Principles of Plant Breeding (1960); Simmonds, N.W., Principles of Crop

Improvement (1979); Sneep, J. et al., (1979) Tomato Breeding (p. 135-171) in:

Breeding of Vegetable Crops, Mark J. Basset, (1986, editor), The Tomato crop: a scientific basis for improvement, by Atherton, J.G. & J. Rudich (editors), Plant

Breeding Perspectives (1986); Fehr, Principles of Cultivar Development — Theory and

Technique (1987).

Also, any plants, plant tissue or organ obtainable according to the above method is encompassed herein. In a preferred embodiment hybrid plants are provided, comprising modified texture characteristics. Such hybrids are for example generated by crossing inbred parental lines and collecting the (hybrid) seeds produced. The parents may for example each be homozygous for the same or for different high expressing alleles, which are then combined in the hybrid seeds and plants.

TILLING and mutagensis It is also an embodiment of the invention to use non-transgenic methods, e.g. mutagenesis systems such as TILLING (Targeting Induced Local Lesions IN Genomics; McCallum et al, 2000, Nat Biotech 18:455, and McCallum et al. 2000, Plant Physiol. 123, 439-442, both incorporated herein by reference) and selection to generate plant lines which produce higher levels of one or more StTLRP proteins and sttlrp mRNA transcripts according to the invention. Without limiting the scope of the invention, it is believed that such plants could comprise point/deletion mutations in the promoter that are binding sites for repressor proteins that would make the host sttlrp gene constitutive or higher in expression. Preferably StTLRP protein levels in the mutant or parts of the mutant are at least about 2, 5, 10, 15% or more increased in the mutant compared to non-mutant plants. TILLING uses traditional chemical mutagenesis (e.g. EMS mutagenesis) followed by high-throughput screening for mutations (e.g. using CeI 1 cleavage of mutant-wildtype DNA heteroduplexes and detection using a sequencing gel system), see e.g. Henikoff et al. Plant Physiology Preview May 21, 2004. Thus, non-transgenic plants, seeds and tissues comprising an enhanced sttlrp gene expression in one or more tissues and comprising one or more of the StTLRP phenotypes according to the invention (modified texture) and methods for generating and identifying such plants is encompassed herein.

The method comprises in one embodiment the steps of mutagenizing plant seeds (e.g. EMS mutagenesis), pooling of plant individuals or DNA, PCR amplification of a region of interest, heteroduplex formation and high-throughput detection, identification of the mutant plant, sequencing of the mutant PCR product. It is understood that other mutagenesis and selection methods may equally be used to generate such mutant plants. Seeds may for example be radiated or chemically treated and the plants screened for a modified StTLRP phenotype. In another embodiment of the invention, the plant materials are natural populations of the species or related species that comprise polymorphisms or variations in DNA sequence at the sttlrp orthologous coding and/or regulatory sequence. Mutations at the sttlrp gene target can be screened for using a ECOTILLING approach (Henikoff et al 2004, supra). In this method natural polymorphisms in breeding lines or related species are screened for by the above described TILLING methodology, in which individual or pools of plants are used for PCR amplification of the sttlrp target, heteroduplex formation and high- throughput analysis. This can be followed up by selecting of individual plants having the required mutation that can be used subsequently in a breeding program to incorporate the desired sttlrp -orthologous allele to develop the cultivar with desired trait.

In a further embodiment non- transgenic mutant plants which produce lower levels of StTLRP protein and mRNA in one or more tissues are provided, or which completely lack StTLRP protein in specific tissues or which produce a non-functional StTLRP protein in certain tissues, e.g. due to mutations in one or more endogenous sttlrp alleles.

For this purpose also methods such as TILLING may be used. Seeds may be mutagenized using e.g. radiation or chemical mutagenesis and mutants may be identified by detection of DNA polymorphisms using for example CEL 1 cleavage.

Non-functional StTLRP alleles may be isolated and sequenced or may be transferred to other plants by breeding methods.

Mutant plants can be distinguished from non-mutants by molecular methods, such as the mutation(s) present in the DNA, StTLRP protein levels, sttlrp RNA levels etc, and by the modified phenotypic characteristics.

The non-transgenic mutants may be homozygous or heterozygous for the mutation conferring the enhanced expression of the endogenous sttlrp gene(s) or for the mutant sttlrp allele(s).

Sequences

SEQ ID NO 1: amino acid sequence of the Solanum tuberosum StTLRP Δ7 protein from parent E. SEQ ID NO 2: amino acid sequence of the Solanum tuberosum StTLRP protein from parent C.

SEQ ID NO 3: amino acid sequence of the Solanum tuberosum StTLRP Δ7 protein from parent E, without the putative secretion signal peptide. SEQ ID NO 4: amino acid sequence of the Solanum tuberosum StTLRP protein from parent C without the putative secretion signal peptide.

SEQ ID NO 5: cDNA sequence of the Solanum tuberosum encoding the StTLRP Δ7 protein from parent E. SEQ ID NO 6: cDNA sequence of the Solanum tuberosum encoding the StTLRP protein from parent C.

SEQ ID NO 7: genomic DNA encoding the StTLRP Δ7 protein from parent E.

SEQ ID NO 8: PCR primer F Taq for qRT-PCR.

SEQ ID NO 9: PCR primer R Taq for qRT-PCR. SEQ ID NO 10: PCR primer Fl; forward primer for genomic/cDNA amplification of sttlrp.

SEQ ID NO 11: PCR primer Rl; reverse primer for genomic/cDNA amplification of sttlrp.

SEQ ID NO 12: PCR primer RΔ7; reverse primer for genomic/cDNA amplification of the Δ7 allele o f sttlrp.

Figure Legends

Figure 1 (A-D) Photographs of steam cooked potato tubers of mealy and non- mealy /firm tubers. Three replicates of steam cooked potato tubers of two representative genotypes (C x E), for either a mealy (A) or a non-mealy (C) cooking type. Enlargements of part of the tuber surface of tubers showing a mealy (C) and a non- mealy (D) phenotype.

Figure 2 Distribution of the number of individuals from a diploid backcross population (CxE) over the different classes of texture after cooking with values ranging from non- mealy/firm (1) to mealy/crumbly (6). Values are the average of three independent replicates. The total number of genotypes selected for bulk segregant analysis (bulk A) for either the mealy or non-mealy bulk are indicated for each of the classes. Figure 3 (A) Sequence alignments of predicted StTLRP protein sequence in potato (S. tuberosum) including, the identified StTLRP protein, the tomato TLRP protein (CAA54561), and TLRP protein predictions of both potato clones C and E. The TLRP protein in the E-parent was identified as an allelic variant and is therefore identified as TLRP Δ7. (B) Graphical overview of StTLRP sequence with predicted start and stop codon and signal peptide indicated with a grey box. Relative positions of primers for amplifying either the entire StTLRP gene (Fl/Rl) or TLRP Δ7 (Fl/R_ Δ7) are indicated. StTLRP specific primers for quantitative RT-PCR analysis are also indicated (F Taq/R Taq). (C) PCR amplification products for StTLRP on C and E genomic or cDNA using the primer combinations as indicated in (B). StTLRP_ Δ7 allele specific amplification bands are indicated with white arrows. For the genomic DNA samples, absence (aa) or presence (ab) of allele TLRP_ Δ7 are indicated.

Figure 4 Expression levels of StTLRP in 56 genotypes of the C x E population including both parental lines, relative to the internal control (ΔCt). Genotypes were plotted starting from the highest expression level detected (ΔCt=O) and positioned accordingly. Presence of the TLRP Δ7 allele was scored for each genotype and indicated as follows: presence (ab) white bar and absence (aa) grey bar. Relative expression levels were averaged from three replicated measurements. Figure 5 Distribution of the number of individuals from the diploid backcross population (C x E) over the different classes of tuber texture after cooking, ranging from firm/non-mealy (1) to mealy/crumbly (6) divided in two groups based on the presence (white bars) or absence (grey bars) of allele TLRP_ Δ7.

Figure 6 Genetic map of chromosome 9 of the E parent (E9) and QTLs for potato tuber texture after cooking and StTLRP expression. (A) Graphical representation of the genetic map of chromosome E9 with distances of the individual markers indicated (cM). For readability additional markers with identical map positions are indicated with an asterisk (*). LOD scores for potato tuber texture after cooking (B) and StTLRP expression levels (C) are plotted relative to the marker positions on chromosome E9 (dashed line). Minimum significant threshold level (LOD 3.0) is indicated with a black line. Predicted inner and outer QTL interval regions are indicated with black bars on the left hand side of the QTL plots.

The following non-limiting Examples describe the use of sttlrp genes for modifying plant phenotypes. Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, and Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.

EXAMPLES

1. Experimental procedures 1.1 Plant Material

The diploid backcross population (C x E) consisting in total of 251 individuals was obtained from the cross between C (USW5337.3) and E (77.2102.37). Clone C is a hybrid between S. phureja and S. tuberosum dihaploid USW42. Clone E is the result of a cross between clone C and the S. vernei-S. tuberosum backcross clone VH34211. Textural changes of tubers after cooking were determined on two consecutive harvests (1998, 1999). Harvested tubers derived from field experiments with three replicates for each genotype, each consisting of two plants. Tubers of the three replicates were harvested and stored for three weeks in controlled conditions before being analyzed. Three tubers of each sample were peeled and steam cooked for 20 minutes after which texture was visually scored on a nominal scale ranging from 1 (firm/non-mealy) to 6 (extreme mealy). A total of 226 genotypes were scored for each of the three harvests and averaged for each year separately. An averaged texture value for each genotype was calculated from the two harvest years and used in QTL analysis. All QTL analysis was performed using the software package MapQTL® 5.0 (Van Ooijen, 2004, MapQTL ® 5, Software for the mapping of quantitative trait loci in experimental populations. Kyazma B.V. Wageningen, The Netherlands). For expression studies, sprouting tubers of 94 CxE individuals, including both parent lines, were potted in five replicates in 5L soil-filled pots in the greenhouse and grown for 3 months. Plants were regularly scored for tuber formation and tubers (>2cm) were harvested from a single plants at around 2¹A week intervals and immediately frozen in liquid N2. A single tuber harvest of a subset of the genotypes, that represent a similar developmental stage having had a period of four weeks of tuber growth, were selected for gene expression studies (56 genotypes).

1.2 RNA-isolation and microarray hybridization Total RNA was isolated from tubers as described by Bachem et al., (1996, supra). mRNA was purified using the GenElute™ mRNA miniprep kit (Sigma Aldrich, Zwijnberg, the Netherlands) and mRNA quality and quantity was checked using the NanoDrop NDlOO (NanoDrop Technologies, Wilmington, Delaware USA). Equal amounts of purified mRNA from the selected individuals were pooled in bulks of ten genotypes for either mealy or non-mealy tuber characteristics, bulk A(m) and bulk A(nm) respectively. Similarly, four bulks of five non-overlapping genotypes were made, bulk B(m), B(nm), C(m) and C(nm). All bulks were prepared in two repeats and first strand cDNA synthesis followed by target labelling with either the Cy3- or Cy5- dye (Amersham BioSciences, Roosendaal, the Netherlands) was performed using the Superscript™ Indirect cDNA Labelling System (Invitrogen, the Netherlands). Expression levels of genes within the selected bulks differing in textural changes after cooking were determined relative to one another in a swap dye experiment using the dedicated potato cDNA-array (Kloosterman et al., 2005, supra). Microarray slides were pre-hybridized and processed as described in van Doom et al., (2003, Plant MoI Biol 53, 845-863). Hybridization of the target samples was performed in the HybArrayl2™ (Perkin Elmer, Niewerkerk, the Netherlands) hybridization station at 42⁰C over a period of 20 hours. Following hybridization, slides were washed as described in van Doom et al. (2003, supra). Slides were immediately scanned using a Scanarray®ExpressHT scanner according to the manufactures specifications (Perkin Elmer, Niewerkerk, the Netherlands). Spotfinding, data extraction, LOWESS normalization and several quality control filters (Spot quality, Low intensity threshold, Signal to Noise Ratio) were performed with the ScanArray® (Perkin Elmer, Niewerkerk, the Netherlands) and the Microsoft® Excel software package. Detection of significantly differentially expressed genes shared between both suppression clones was performed using the SAM software (Tusher et al., 2001, Proc Natl Acad Sci U S A 98, 5116-5121) with a set FDR of 0 and minimal 2-fold expression difference in all three bulks analyzed. The identified differentially expressed gene set was imported into the SPSS software package and were subjected to T-testing (p<0.005) for significant expression change >2-fold in both directions.

1.3 DNA Sequence analysis The full length sequence of the potato StTLRP was obtained by sequencing cDNA clone BG096637 deriving from a potato leaf EST library (cv. Kennebec) using vector based primers T3 and T7. All sequence data was analyzed with the Vector NTI suite (Informax, Bethesda, USA). Homology searches were carried out using the blastX program (Altschul et al, 1997, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402). Protein alignments were carried out using the ClustalX program (Thompson et al., 1997, Nucleic Acids Res 25, 4876-4882).

1.4 Quantitative RT-PCR

Relative expression levels of the StTLRP gene were determined by real-time quantitative reverse transcriptase PCR (qRT-PCR) on a Perkin Elmer Abi Prism 7700 Sequence detector (Perkin Elmer, Niewerkerk, the Netherlands) following the protocol described in Kloosterman et al., (2005, supra). Potato ubiquitin primers (ubi3) were used as a control. Relative quantification of the target RNA expression level and standard deviation was performed using the comparative Ct method according to the User Bulletin #2 (ABI Prism 7700 Sequence Detection System, December 1997, Applied Biosystems). The primer sequences for the genes studied are as follows: F Taq forward primer 5 '-TCCAATGCGGTAAACGTTGA-S ', R-Taq reverse primer 5 '-CTTACCATAGCCGCCACCAT-3 ' and ubi3 (L22576) forward primer 5'- TTCCGACACCATCGACAATGT-3', reverse primer 5 '-CGACCATCCTCAAGCTGCTT-S '.

1.5 Genetic map and QTL analysis To score presence of allele StTLRP_Δ7 within the C x E population including both parents (C and E), PCR was performed on genomic DNA and cDNA using the following primer sequences:

Fl: 5 '-ATGGGTTCCAAGGCAATTATGTT-S ',

Rl: 5 '-GAATGGCTTTATTCATACTTGTT-S ', RΔ7: 5 '-GCAGCAGTATTTTTTGTGGCAT-S '.

Using primers Fl and Rl, 209 genotypes were scored for presence (ab) or absence (aa) of allele TLRP Δ7. Integration of StTLRP marker in the existing genetic map of C x E (Celis-Gamboa, 2002, The life cycle of the potato (Solanum tuberosum L.): from crop physiology to genetics. PhD thesis. Wageningen: Wageningen University) was performed using the software program JoinMap 3.0 (Van Ooijen and Voorrips, 2001, Joinmap® 3.0, Software for the calculation of genetic linkage maps. Plant Research International Wageningen, The Netherlands).

2. Results

2.1 Distribution of potato tuber texture after cooking scores

The offspring of a diploid backcross between diploid parents C and E exhibits strong segregation for a large number of tuber quality traits including potato tuber cooking type. Textural changes of tubers after cooking (i.e. cooking type) from individuals of the population were determined in two consecutive years. Texture of steam cooked potato tubers was visually scored and categorized on a nominal scale ranging from firm/non-mealy (1) to extreme mealy tubers (6).

Figure 1 shows an example of the differences that could be observed in the texture of potato tubers within the CxE population after cooking, including a mealy (Figure IA) and a firm/non-mealy tuber genotype (Figure 1C). The outer cell layers appear to be shed off (sloughing) and the loose layers can be typified as slurry consisting of intact individual cells as shown in the enlargement of part of the tuber surface (Figure IB). Non-mealy tubers maintain a compact appearance having a relatively firm and glossy surface (Figure ID). Cross-sections of these tubers reveal similar pheno types in the inner parts of the tuber although in general less severe as observed in the outer layers. Potato tuber cooking type as a quantitative trait shows a high level of heritability (h2) in both harvest years (1998; 0.95 and 1999; 0.80), indicating the observed variance in both years is primarily due to genotypic variation present within the population. In addition, a strong correlation (0.85) was found between the individual datasets of the two harvest years and average texture scores were calculated for 226 genotypes by combining the two datasets. The distribution of the number of plants over the different texture classes is shown in Figure 2. Potato tuber cooking type within the CxE population shows a transgressive segregation in which both parental clones exhibit a relatively firm/non-mealy texture profile (1.0 and 1.8 for the C and E parent, respectively). 2.2 Bulked expression analysis for tuber texture after cooking

To identify candidate genes involved in the determination of texture after cooking we analyzed gene expression within a subset of genotypes from the CxE population. In a large scale greenhouse experiment 90 individuals of the CxE population, segregating for a number of different tuber quality traits, were grown in five repeats and regularly scored for tuber formation and harvested at different intervals. One of the most predominant phenotypes within the CxE population is the difference in earliness resulting in different time points of tuber formation throughout the growing season (Celis-Gamboa et al, 2003, Annals of Applied Biology 143, 175-186). To circumvent the problem of analyzing gene expression in tubers that represent differences in tuber physiological age and therefore metabolic status, only significantly sized (>2cm) tubers from plants that have had at least a four week period of tuber growth were used. In addition, a pooling strategy was implemented to further reduce the variability between individual genotypes based on their different plant stature or other tuber qualities allowing the detection of differentially expressed genes that are associated with after cooking texture characteristics. For expression analysis, mRNA from harvested tubers of ten individuals at both ends of the texture after cooking type spectrum were pooled in two bulks representing either a mealy or a non-mealy tuber cooking type (Figure 2). The two bulks, for either a mealy (m) or non-mealy (nm) cooking type were named bulk A(m) and A(nm) respectively, and the number of individuals from each of the different texture classes selected for either bulk are indicated in Figure 2.

To allow the detection of more subtle changes in expression levels we reduced the initial bulk size and divided bulk A(m) and A(nm), each containing ten individuals, into two separate pools of five non-overlapping individuals into bulk B(m) and B(nm) and bulk C(m) and C(nm), respectively. Using a dedicated potato cDNA microarray, specifically designed for studying tuber development and tuber quality traits and containing a large number of cell wall synthesis and maintenance genes, relative expression levels were obtained by hybridizing the labelled target samples representing the mealy bulks (A(m), B(m) and C(m)) against the corresponding non-mealy bulks (A(nm), B(nm) and C(nm)). For all hybridizations a swap-dye experiment was included and obtained expression data was normalized and analyzed as described in Experimental procedures. Genes that were consistently and significantly higher expressed (>2-fold) in either the mealy or non-mealy tuber bulks are listed in Table 2.

Only three genes showed consistent differential expression patterns across the three bulks indicating a putative correlation between texture after cooking and the level of gene expression. From these, only a single candidate gene showed an on average higher expression in the non-mealy bulks in comparison to the mealy bulks. The differentially expressed gene, represented by cDNA clone (BG096637) exhibited strong sequence similarity to a tomato extra cellular cell wall protein (TLRP). Interestingly, the observed differential expression of the gene is much larger (25 -fold) in bulks C in comparison to the expression ratio's found in bulks A and B (2.2 and 2.5-fold, respectively). This observation may indicate that the genotypes represented in both the mealy and non-mealy C bulks (C(m) and C(nm)) have either a more similar and consistent high or low expression level, in comparison to the other bulks, increasing the expression difference.

Table 2: Genes with significant differential expression between contrasting bulks for potato tuber cooking type cDNA clone

Median¹ Fold-chanj *e across bulks GenBank Functional Homology accessions A² B³ C⁴

BG096637 Tomato extra cellular matrix protein 2.2 2.5 25.1 (TLRP)

BE919835 chlorophyll a/b binding protein type I -3.4 -4.9 -3.2

BE920360 chlorophyll a/b binding protein type I -2.6 -3.1 -2.6

Median values were calculated from 2 independent hybridizations including a swap dye and replicates on the array.

Fold difference in relative gene expression levels between bulks A(m) and A(nm) each containing ten individuals from the CxE population exhibiting either a mealy (m) or non-mealy (nm) cooking type

Fold difference in relative gene expression levels between bulks B(m) and B(nm) each containing 5 individuals that were also part of the A bulks

⁴ Fold difference in relative expression levels between bulks C(m) and C(nm) each containing 5 different genotypes that were part of the mealy and non-mealy A bulks but non-overlapping with the individuals selected for the B bulks The two other differentially expressed genes (BE919835, BE920360), with sequence homology to chlorophyll a/b binding proteins (CAB), show on average a higher expression in the mealy bulks. However, based on their putative function and the finding that both these genes were also found to be differentially expressed in bulks for other unrelated tuber quality traits (data not shown), they are considered to be false positives as a result of very strong expression in only a small number of individuals that skew the average expression levels within the bulk. A likely explanation for the identification of this false positive, might be the growth conditions, since the potato plants were grown in pots, tuber formation occurs relatively close to the surface and a small subset of the harvested tubers may have been directly exposed to light, inducing strong expression of the genes that are part of the light harvesting complex.

It is well documented that the cell wall has an important role in determining potato tuber texture characteristics, and therefore, the identification of a potato EST with high sequence similarity to an extra cellular cell wall protein, that is more highly expressed in the tuber bulk with a more firm/non-mealy tuber texture, was considered to be a promising candidate gene and was analyzed in greater detail.

2.3 Sequence analysis and identification of an allelic variant of StTLRP

The full length sequence of the identified candidate gene was obtained by sequencing EST clone (BG096637) derived from a leaf cDNA library of potato var. Kennebec. Sequence analysis revealed an open reading frame (ORF) of 237 nucleotides with a predicted protein of 78 amino acids, which showed some sequence similarity with the tomato (S. lycopersicum) tyrosine and lysine rich protein (TLRP; X77373).

The predicted potato protein is characterized by a high level of tyrosine (7) and lysine (7) residues as well as the presence of a highly conserved N-terminus signal peptide targeting the protein to the extra cellular cell wall matrix (Figure 3A). Based on these observations, the identified gene was designated StTLRP.

The new potato TLRP protein is smaller than the tomato TLRP due to the absence of two stretches of 3 and 7 amino acids, the latter disrupting a potential Cys domain (CD) that was identified in a tobacco TLRP protein (NtTLRP; CAB67122), and thought to be involved in cross-linking soluble proteins to the cell wall making them insoluble (Domingo et al, 1999, Plant J 20, 563-570).

To allow the design of StTLRP specific primers for qRT-PCR analysis of individuals of the CxE population, we first needed to obtain the StTLRP sequences of both parental clones to account for any allelelic variations. Using StTLRP specific primers (Fl/Rl), single bands were amplified for both parents using cDNA templates obtained from C and E growing tubers (Figure 3C). The amplified product in the C parent was slightly larger (372bp) in comparison to the E-parent (348bp). Sequence analysis of the amplified PCR product in the E-parent revealed a predicted ORF and protein sequence that was identical to the identified StTLRP (78 amino acids). Interestingly, within the slightly larger amplified PCR product of the C-parent two different sequences could be identified, having few nucleotide substitutions in the 5'UTR, however, giving rise to identical predicted protein sequences of 85 amino acids in length.

Sequence alignment of the predicted protein sequences of both parental lines show that the larger sequence found in the C-parent is the result of the presence of an additional 7 amino acids stretch identical to the sequence present in the tomato TLRP (Figure 3A). To further investigate this difference in amplification products in both parental lines, specific primers (F1/RΔ7) were designed to only amplify the TLRP sequence containing the 7 amino acid deletion site (TLRP Δ7). An amplified product using these gene specific primers was only detected in the E parent in both genomic (Figure 3C) and cDNA templates (data not shown), suggesting the identified StTLRP gene is actually an allelic variant with a 7 amino acid deletion and is hereafter referred to as TLRP Δ7. Using the StTLRP primers (Fl/Rl) on genomic DNA, a sequence region spanning the entire ORF and parts of the 3'UTR was amplified from genomic DNA of both parental clones (Figure 3B,C) . Here, two amplified bands (989bp and 852bp) were detected in the E parent while only a single amplification band (989bp) was found in the C parent. Sequence analysis revealed that the lower band (852bp) within the E parent corresponded to the identified allele, TLRP Δ7, which showed an additional deletion site of around 115bp present within the identified intron of the StTLRP genomic sequence. Based on a conserved region of the StTLRP gene in the C and E lines, primers were designed (Figure 3B; F Taq/R Taq) to measure expression of StTLRP in tubers of CxE genotypes with quantitative RT-PCR. Gene expression levels of 56 genotypes, including the individuals represented in the bulks, were measured and showed remarkable variation relative to the internal control (Figure 4). More specifically, all genotypes harbouring the TLRP Δ7 allele, showed on average a much higher level of expression in comparison to genotypes lacking the respective allele. Both parental lines show a large difference in expression levels, E has a ΔCt of 0.11 and C a ΔCt of 6.11 which translates to a (2-ΔΔCt) 64-fold higher expression of StTLRP in the E-clone. Despite the observed differences in expression levels of StTLRP in both parental lines, with a higher expression found in E, both exhibit a relatively non-mealy texture profile indicating that other factors or genes are likely to be involved in regulating potato tuber cooking type within the CxE population. To further investigate a possible correlation between presence or absence of the TLRP Δ7 allele and potato tuber cooking type, the distribution of the individual genotypes over the different texture classes was re- examined based on TLRP Δ7 scores (Figure 5). In general, genotypes harbouring the TLRP Δ7 allele appear to have a more firm/non-mealy texture profile while the distribution of the genotypes lacking the TLRP Δ7 allele, shows a shift towards a more mealy tuber texture. However, a substantial number of genotypes lacking the TLRP Δ7 allele do exhibit a firm/non-mealy texture indicating that the presence of TLRP Δ7 is not strictly required for producing a firm/non-mealy phenotype. In fact, the opposite may be true, in which absence of the TLRP Δ7 allele is required to produce a mealy phenotype, since it was observed that 34 of the 40 mealiest genotypes lack the identified TLRP Δ7 allele.

2.4 QTL analysis of potato tuber cooking type and StTLRP expression The identified allelic variant of StTLRP present in the E-parent and absent in the C- parent, shows a clear segregation in the offspring in a cross between C and E. Presence (ab) or absence (aa) of TLRP Δ7 was scored within 209 individuals resulting in a 114 (ab) : 95 (aa) ratio resembling the expected 1:1 Mendelian segregation. Allelic scores were used to integrate the StTLRP marker into an existing genetic map of CxE (Celis- Gamboa, 2002, supra) and was subsequently mapped on the long arm of chromosome 9 of the E-parent (48cM) and positioned between markers E32M49-168e9 (37.8cM) and E32M48-274.9e9 (53.OcM) (Figure 6A).

To examine a possible correlation between StTLRP expression levels and tuber texture after cooking, QTL analysis on the texture data and expression levels of candidate gene StTLRP was performed. Firstly, interval mapping using the average texture data over the two harvest years of 226 genotypes yielded a single significant QTL (LOD 7.51) on the long arm of chromosome E9 as shown in Figure 6B.

The predicted QTL on chromosome 9 of the E-clone explained 14.3% of the observed variance within the combined harvest years. Genome wide significant thresholds for maximum LOD scores were calculated after performing a permutation test and was set at a LOD of 3.0 (p=0.05). The texture QTL shows the highest association with the StTLRP marker in the non-parametric Kruskal-Wallis test (K*= 29.451 with ldf and p- value < 0.0001).

Secondly, the relative expression levels of the identified StTLRP gene in potato tubers of 56 individuals was treated as a quantitative trait and interval mapping produced a single strong expression QTL (eQTL; LOD 21.6) positioned directly above the StTLRP marker on the genetic map. (Figure 6C). The high association of StTLRP expression levels with the StTLRP genetic marker (K*= 39.55 with ldf and p-value 0.0001) on the same chromosomal location, strongly indicates czs-acting transcriptional regulation of StTLRP. Moreover, the identified eQTL for StTLRP co-localizes with the QTL for texture after cooking providing strong evidence for the association of StTLRP expression levels or allelic variation with the texture of cooked potatoes.

2.5 Discussion

In this study we have implemented a genetical genomics approach in an attempt to identify novel candidate genes for textural changes in steam cooked potato tubers by using microarray technology. Since hybridization of each individual of the CxE population on a microarray would be very costly and time consuming we have pooled individuals to reduce the number of hybridizations as suggested by Jansen and Nap (2001, supra) and Kerr (2003, Biometrics 59, 822-828). The efficiency and reliability of using a pooling strategy for studying gene expression has been tested in a number of studies (Agrawal et al., 2002, J Natl Cancer Inst 94, 513-521.; Kendziorski et al, 2003 Biostatistics 4, 465-477; Glass et al., 2005, Biosci Biotechnol Biochem 69, 1098-1103) and has shown to be a valid method for reducing the number of microarray hybridizations, however their experiments did not encompass segregating plant populations. Pooling of genotypes that share a phenotype but are otherwise genetically variable masks expression differences that may be a result of environmental conditions or other variables. The selection of individuals for bulks exhibiting extreme phenotypes unrelated to the trait of interest may, however, result in the identification of false positives. Hence, choice of bulk size is vital when attempting to minimize the number of false positives without loosing too much sensitivity in order to allow detection of more subtle changes in gene expression. Furthermore, the best time point for harvesting and analyzing gene expression in potato tubers is difficult to determine if one does not know at which developmental stage a quality trait, like cooking type, is being established. In a previous study we have shown that gene expression is highly variable during the initial stages of potato tuber development and often stabilizes during the stages of tuber filling before entering a state of dormancy (Kloosterman et al., 2005, supra). In a pilot study of 8 individuals of the CxE population, gene expression was measured for each individual on two separate time points representing actively growing tubers and fully matured dormant tubers. Besides a much larger percentage of genes on the array being more highly expressed in the active growing tubers in comparison to the dormant tubers, the level of expression variance was also much larger in the former (Vorst et al., unpublished results). Therefore, we have chosen to study gene expression in tubers of a similar developmental stage that are however still active in their tuber filling stage, allowing the detection of a large number of expressed genes. For other quality traits however, one could decide to study either dormant tubers or younger tubers if expected expression differences at these stages would be vital in determining a particular quality trait.

Based on the results from our microarray experiments we have identified a new gene (StTLRP) for involvement in the observed textural changes in tubers after cooking. We found a strong correlation between expression levels of the this gene (StTLRP) with potato tuber cooking type as exemplified by the co-localization of the eQTL and the QTL for texture on chromosome E9 (Figure 6A-C). In addition, sequence analysis allowed the identification of an allelic member of StTLRP that was shown to be responsible for the high expression levels and was mapped to the same genetic map location (Figure 6A). The observation that the StTLRP genetic map position co- localizes with the identified expression QTL indicates czs-acting transcriptional control. The precise mechanism controlling the level of StTLRP expression is however still unknown.

Interestingly, the identified StTLRP lacks a stretch of 7 amino acids in comparison to the tomato TLRP disrupting a likely CD domain thereby potentially altering StTLRP protein conformation and capacity to bind previously insoluble proteins to the cell wall (Figure 3A). The presence of this deletion site within a potential CD domain, appears to be an allelic variant within the CxE population giving rise to genetic and potentially phenotypic variability.

There appears to be a stronger association between the absence of the high expresser allele TLRP Δ7 in extreme mealy tubers than presence of the same allele in firm/non- mealy tubers (Figure 5). This observation indicates that the other non- identified StTLRP allele(s) coming from the C and E parent may also play a role in the observed mealy tuber textures. Genomic sequence analysis of amplified PCR products using StTLRP specific primers as well as Southern blotting with StTLRP cDNA as probe, have shown that the identified StTLRP is most likely part of a gene family with highly homologous members, making it difficult to identify the other allelic constituents (Kloosterman, data not shown). Nevertheless, the identification of other alleles present within the CxE population should provide a better picture of the genetic components underlying StTLRP expression and function in relation to potato tuber cooking type.

Claims

1. A method for modulating texture characteristics of plant tissues or plant organs comprising increasing and/or decreasing the mRNA expression levels of a nucleic acid sequence encoding a cell wall protein in said plant tissues or organs, characterized in that said cell wall protein comprises at least 80% amino acid sequence identity over the entire length to SEQ ID NO: 1 or SEQ ID NO: 2.

2. The method according to claim 1, wherein said texture characteristics are firmness and/or mealiness of the plant tissues or organs and wherein said texture characteristics are preferably determined after heat treatment, such as cooking or steaming.

3. The method according to claim 1 or 2, wherein said plant tissues or organs are underground tissues or organs of root and tuber vegetables, preferably potato tubers, sweet potato tubers, yams, cassava, sugar beet or carrot, or fleshy fruit, preferably tomato, apple, pear, banana, peach, nectarine or melon.

4. The method according to claim 3, wherein the cooking-type of said tissue or organ is altered as a result of said modulated mRNA expression levels, preferably from mealy to firm.

5. The method according to any one of claims 1 to 4, wherein said mRNA expression levels are modulated by:

(a) marker assisted selection of one or more alleles of a nucleic acid sequence encoding a cell wall protein comprising at least 80% amino acid sequence identity over the entire length to SEQ ID NO: 1 or SEQ ID NO: 2; or

(b) generating a transgenic plant comprising, integrated in its genome, a nucleic acid sequence encoding said cell wall protein, operably linked to a transcription regulatory sequence active in plant cells.

6. The method according to claim 5, wherein said mRNA expression levels are increased by marker assisted selection in step (a) comprises the steps of: (a) identifying one or more alleles of said nucleic acid sequence, preferably alleles which have a high mRNA expression level;

(b) developing molecular markers which are specific for said alleles;

(c) using said molecular markers to transfer and/or combine one or more of said alleles to/in progeny of a cross between at least one parent plant comprising said allele and another plant; and optionally

(d) testing the mRNA expression level and/or the texture characteristics of the progeny of said cross at one or more times; and

(e) identifying progeny comprising said alleles and/or allele combinations and preferably firm tissue texture characteristics.

7. The method according to claim 6, wherein said allele encodes a cell wall protein comprising a cysteine domain having the sequence C (X)_2-3 (C) I_-2 (X)_4-6 (C)_0-1 (X)_0-3 CC.

8. The method according to claim 6 or 7, wherein said molecular markers are one or more PCR primers or nucleic acid probes comprising a contiguous stretch of at least 10 nucleotides of a nucleic acid sequence comprising at least 80% sequence identity over the entire length to any one of the nucleic acid sequences of SEQ ID NO: 5 to SEQ ID NO: 7, or to a nucleic acid sequence genetically linked in cis to said nucleic acid sequence.

9. A plant, plant tissue or organ obtainable according to any one of claims 1 to 8.

10. The plant, plant tissue or organ according to claim 9 belonging to the genus Solarium, Ipomoea, Dioscorea or Manihot.

11. A transgenic plant or plant tissue or organ comprising integrated in its genome a chimeric gene, characterized by said chimeric gene comprising a transcription regulatory sequence active in plant cells operably linked to a nucleic acid sequence encoding a cell wall protein, wherein said cell wall protein comprises at least 80% sequence identity to SEQ ID NO: 1 and/or SEQ ID NO: 2.

12. Use of a nucleic acid sequence for the modulation of texture characteristics of plant tissues or organs, wherein said nucleic acid sequence encodes a protein comprising at least 80% sequence identity over the entire length to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

13. A chimeric gene or vector comprising a nucleotide sequence encoding a protein comprising at least 80% sequence identity over the entire length to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

14. A protein comprising at least 80% sequence identity over the entire length to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.