CYTOKININ RIBOSIDE PHOSPHORYLASE FROM POTATO AND ITS USE

Field of Invention

The present invention relates to the provision of tuber- producing plants in which the level of tuberising activity or tuber germination is altered relative to that of conventional tuber-producing plants. In particular, the invention relates to the provision of methods of obtaining transformed tuber-producing plants comprising nucleic acid sequences that effect altered tuberisation patterns from tuber precursor structures such as the stolon tip in stolon-producing plants or altered patterns of sprouting from dormant shoot buds on tubers at germination, transformed plant cells produced by such methods and transformed plants derived therefrom, nucleic acid sequences, vectors and constructs therefore, proteins, and uses thereof.

Background of Invention

Plants that are capable of giving rise to tuberous structures typically comprise foodstuff storage organs that have at least one apex or node located on the storage organ from which shoot growth is initiated. Plants that produce sprouting shoot structures from storage organs may

comprise, for example, two or more apices or nodes that may be found on the storage organs from which shoots capable of providing rooting and shooting tips are able to grow: such storage organ structures may give rise to rooting systems and/or aerial parts. Such shooting and rooting cells, such as growing cells located in the tip of the sprout, may typically use energy that is derived from a storage organ. Each storage organ is typically able to give rise to an independent plant which is able to grow aerial shooting and sub-surface rooting parts which in turn are able to give rise to new storage organs and so new plants. In certain plants the storage organ comprises a foodstuff store or reserve within a swollen axis, and is referred to as a stem tuber, for example a potato tuber, or a tuberous root (sometimes referred to as a ^λ tuber' , e.g. a dahlia ^λ tuber' ) or other term. The ^λ foodstuff store' of a plant typically comprises carbohydrate, such as starch (e.g. in potato), or glycans, such as fructan (e.g. in Jerusalem artichoke), in addition to storage proteins (such as patatin in potato) and minerals that are typically present in significant amounts in the storage organ.

Commercial growers of tuberous plants as defined herein face at least two problems with respect to a) the storage

of so-called "seed" tubers and b) the propagation of new plants from tubers. The first problem relates to the numbers of tubers that any one plant may produce in a season. It is desirable that plants capable of growing tubers as defined herein produce as many viable tubers as possible for tuber multiplication purposes, and hence "seed" tuber sales to growers. The second problem relates to undesirable germination of tubers during storage. Such tuber germination activity may have a detrimental effect on the shelf life of tubers and can result in economic losses that occur as a result of premature sprouting from the tuber, and hence reduced seed tuber sales, or indeed, reduced sales of tubers destined for food sales.

Summary of Invention

It has now been found by the present inventors that tuberisation of the stolon structure can be altered or regulated in stolon-producing plants of the invention.

It is an object of the present invention to alleviate and/or obviate problems associated with the storage and/or generation of tubers on tuber-producing plants.

According to the present invention there is provided a method of altering the expression of at least a heterologous or exogenous cytokinin riboside phosphorylase in a plant cell of a tuber-producing plant that comprises: 1) introducing into the said plant cell a first isolated cytokinin riboside phosphorylase nucleic acid sequence operably linked to an exogenous promoter that drives expression in a plant cell wherein the said first isolated nucleic acid sequence comprises a promoter operably linked to a nucleic acid sequence that encodes at least an heterologous or exogenous cytokinin riboside phosphorylase sequence ; and

2) generating from the said first isolated cytokinin riboside phosphorylase nucleic acid sequence an heterologous or exogenous cytokinin riboside phosphorylase mRNA sequence.

In a variant of the above method there is provided a method of producing at least a heterologous or exogenous cytokinin riboside phosphorylase in a plant cell of a tuber-producing plant that comprises:

1) introducing into the said plant cell a first isolated cytokinin riboside phosphorylase nucleic acid sequence operably linked to an exogenous promoter that drives

expression in a plant cell wherein the said first isolated nucleic acid sequence comprises a promoter operably linked to a nucleic acid sequence that encodes at least an heterologous or exogenous cytokinin riboside phosphorylase sequence; and

2) expressing from the said first isolated cytokinin riboside phosphorylase nucleic acid sequence an heterologous or exogenous cytokinin riboside phosphorylase therefrom.

According to a further aspect of the present invention there is provided an isolated nucleotide sequence encoding an antisense RNA molecule complementary to a sense mRNA molecule encoding for a protein having cytokinin riboside phosphorylase activity in the cells of a tuber-producing plant, which nucleotide sequence is under transcriptional control of a promoter and a terminator, both promoter and terminator being capable of functioning in plant cells. Typically, the antisense sequence of the invention is located in the stolon, shoot tip, shoot bud, root, or tuber cells of a tuber-producing plant of the invention. Preferably, the cytokinin phosphorylase activity is located in the stolon tip(s) of a stolon-producing plant. It is to be understood that the terms "stolon tip" and "stolon tips"

are used interchangeably and both forms may be taken to be in the plural or in the singular depending on the context in which these terms are used.

For the purposes of the present invention the term "tuber" as used herein is employed to encompass all expandable storage structures from which shoot structures can be initiated and/or are able to grow. Thus, "tubers" of the invention include stem tubers, tuberous roots, rhizomes, bulbs and corms . Each storage organ of a plant of the invention should be capable of giving rise to one or more tuber precursor structures which, on appropriate plant hormone signalling, is capable of swelling or expanding to form a separate tuber, tuberous root, rhizome, bulb, or corm depending on plant type. From the foregoing discussion it is clear that ^λ tuber-producing plants' and ^tuberous plants' of the invention may give rise to or comprise storage organs as alluded to herein.

Commercial growers of plants that rely on storage organs for the provision of new plants typically harvest and store viable plant material in the form of the storage organs. Such viable plant material takes the form of seed tubers, tuberous roots, bulbs, corms or rhizomes which may be used for the planting of crops, e.g. potato crops or indeed,

commercial growers may also provide crop plant tubers for human or animal feed. Such plants include varieties of Solanum tuberosum (potato) , Dioscorea spp (yams) , Ipomoea batatas (sweet potato) , Helianthus tuberosum (Jerusalem artichoke) , Manihot esculenta (cassava, manioc) ,

Pachyrhizus erosus (jicama), Xanthosoma spp (malanga) , Colocasia esculenta (taro) , Eleocharis dulcis (water chestnuts) , Oxalis tuberosa (oca) , Maranta arundinacea (arrowroot) , Allium oschaninii (shallot) , A. sativum (garlic), A. schoenoprasum (chives) and the like. Certain ornamental plants may also produce tubers of the invention as defined herein, such as varieties of tulip, amaryllis, hyacinth, daffodil, narcissus, cyclamen, lily, lily of the valley, iris, gladiolus, crocus, crocosmia, snowdrop, bluebell, dahlia, freesia, gloxinia, anemone, fritillaria, alstromeria ligtu and hybrids thereof, camassia esculenta, arum italicum, muscari, agapanthus, begonia, acidanthera, ranunculus, ornamental allium and the like. The online Sutton Seeds catalogue 2007 herein incorporated by reference provides examples of many varieties of ornamental plant which produce stolon structures.

The nucleotide sequence encoding the antisense RNA molecule can be of any length provided that the antisense RNA

molecule transcribable therefrom is sufficiently long so as to be able to form a complex with a sense mRNA molecule encoding for a protein having a cytokinin riboside phosphorylase enzymic activity in a tuberising structure, such as a stolon tip plant cell. A suitable example of a protein having cytokinin riboside phosphorylase enzyme activity of the invention is the Solanum tuberosum adenosine/cytokinin riboside phosphorylase 1 (StCKPl). Other proteins that may also have appropriate cytokinin riboside phosphorylase activity of use in the present invention are encoded by Arabidopsis thaliana genes At4g28940 and At4g24350, poplar genes BSP and win4, and the rice genes Os05g0228000 and Os01g0229800. Thus, it is thought that the antisense RNA molecule or short sequences thereof derived from a cytokinin phosphorylase of the invention in planta, in vivo, or in vitro, such as short interfering RNA (siRNA) , form complexes that are capable of interfering with the mRNA of the cytokinin riboside phosphorylase of interest, such as a cytokinin riboside phosphorylase that is involved in stolon tuberisation . Thus, the synthesis of functional protein (s) having tuberisation activity is prevented or substantially inhibited. As a consequence of the interference of the antisense RNA, enzyme activity of cytokinin riboside

phosphorylase protein (s), such as StCPKl, tuberisation from the stolon tip is decreased. Thus, without the intention of being bound by theory it is thought that the antisense RNA molecule complexes with the mRNA of the protein and prevents or substantially inhibits the synthesis of functional protein (s) having stolon tuberisation activity in the stolon tip. As a consequence of the interference of the antisense RNA, enzyme activity of protein (s) involved in cytokinin riboside phosphorolysis in the stolon tip that normally results in tuberisation is decreased, and the effect is that the numbers of tubers that are produced by stolon-producing plants of the invention from the stolon is low relative to the numbers of tubers produced by the stolons of conventional plants of the same species. For the purposes of the description "nucleotide sequence" will be referred to as DNA unless there is different indication. The DNA encoding the antisense RNA can be from about 20 nucleotides in length up to the length of the relevant mRNA produced by the cell. Preferably, the length of the DNA encoding the antisense RNA will be from 20 to 1500 nucleotides in length. When the interfering antisense RNA is interfering siRNA, the length of the siRNA strand is from 20 to 30 nucleotides in length and may be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotide bases in

length. The preferred source of antisense RNA for DNA constructs of the present invention is DNA showing substantial identity or similarity to the genes or fragments thereof of proteins having enzymic activity involved in cytokinin riboside phosphorylase activity, for example StCKPl activity, in the stolon tips, shoot tips, buds or roots of tuber-producing plants. Thus the encoding DNA of constructs of the present invention may be for a cytokinin riboside phosphorylase or fragments thereof such as enzymically active fragments thereof that are capable of causing phosphorolysis of the cytokinin ribosides in the stolon tip.

The invention still further provides a nucleotide sequence which is similar to the above disclosed antisense RNA sequences. By "similar" is meant a test sequence which is capable of hybridising to a sequence which is complementary to a nucleotide sequence of the invention, such as a StCKPl RNA nucleotide sequence. When the test and inventive sequences are double stranded the nucleic acid constituting the test sequence preferably has a Tm within 2O ⁰ C of that of the inventive sequence. In the case that the test and inventive sequences are mixed together and denatured simultaneously, the Tm values of the sequences are

preferably within 1O ⁰ C of each other. More preferably, the hybridization is performed under stringent conditions, with either the test or inventive nucleic acid preferably being supported. Thus either a denatured test or inventive sequence is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of between 5O ⁰ C and 7O ⁰ C in double strength SSC (2x NaCl 17.5 g/1 and sodium citrate (SC) at 8.8 g/1) buffered saline containing 0.1% sodium dodecyl sulphate (SDS) followed by rinsing of the support at the same temperature but with a buffer having a reduced SSC concentration. Depending upon the degree of stringency required, and thus the degree of similarity of the sequences, such reduced concentration buffers are typically single strength SSC containing 0.1% SDS, half strength SSC containing 0.1% SDS and one tenth strength SSC containing 0.1% SDS. Sequences having the greatest degree of similarity are those the hybridization of which is least affected by washing in buffers of reduced concentration. It is most preferred that the test and inventive sequences are so similar that the hybridization between them is substantially unaffected by washing or incubation in one tenth strength sodium citrate buffer containing 0.1% SDS.

The invention still further provides a nucleotide sequence which is complementary to one which hybridizes under stringent conditions with the above disclosed nucleotide sequences .

The skilled addressee will appreciate that nucleotide sequences of the invention as defined herein may be introduced to plant cell genomes of tuber-producing plants. Such introduced antisense nucleotide sequences of the invention when expressed in at least the stolon tip, shoot tip, shoot bud or root help to reduce tuberisation in plants genetically transformed with a cytokinin riboside phosphorylase nucleic acid sequence of the invention.

The invention still further provides a method of inhibiting the production of at least one cytokinin riboside phosphorylase enzyme in a eukaryotic cell, comprising introducing into the said cell a nucleotide sequence comprising a transcriptional regulatory sequence and a sequence contiguous therewith and under the transcriptional control thereof, which contiguous sequence encodes an RNA which consists of at least one single subsequence characterized in that the at least one subsequence has an antisense RNA to an mRNA of a protein having a cytokinin

riboside phosphorylase enzymic activity in the stolon tip(s), shoot tip(s), bud(s) or root(s) of a tuber- producing plant of the invention.

Examples of nucleotide sequences of the invention are provided below. These examples relate to the production of tuberous plants of Solanum tuberosum, Dioscorea rotundata and Ipomoea batatas.

1. The nucleotide sequence of the invention may encode an mRNA which consists--in the 5' to 3' direction--of (i) a promoter, (ii) at least one cDNA of a cytokinin riboside phosphorylase enzyme, such as StCPKl, in reverse orientation i.e. 3' to 5' orientation, (III) a terminator, (iv) optionally a further promoter, (v) the coding region of an antibiotic resistance marker gene such as the HPT II gene (hygromycin) and (vi) optionally a further stop codon. When such a sequence is introduced into the cells of Solanum tuberosum, Dioscorea rotundata or Ipomoea batatas plants, the sequence encoding the mRNA is transcribed. The region of the thus transcribed mRNA which encodes the HPT II gene is translated, whilst the region of the mRNA which encodes the cDNA is not.

2. The nucleotide sequence of the invention may encode an mRNA which consists--in the 5' to 3' direction--of (i) a promoter, (ii) the coding region of an antibiotic resistance marker gene such as the HPT II gene, (iii) a translation stop codon, (iv) optionally a further start codon, (v) a region encoding at least one cDNA of a cytokinin riboside phosphorylase enzyme, for example StCKPl, in reverse orientation i.e. 3' to 5' orientation and (vi) optionally a further stop codon. When such a sequence is introduced into the cells of Solanum tuberosum, Dioscorea rotundata or Ipomoea batatas, the sequence encoding the mRNA is transcribed. The region of the thus transcribed mRNA which encodes the HPT II gene is translated, whilst the region of the mRNA which encodes the cDNA in reverse orientation i.e. 3' to 5' orientation is not translated.

As a further aspect of the invention there is provided a nucleotide sequence capable of differential expression of introduced nucleotide sequences of the invention which comprises a first nucleic acid subsequence encoding a functional cytokinin riboside phosphorylase of the invention in the sense orientation, for example, StCKPl cDNA in the 5' to 3' direction, and at least a second

nucleic acid subsequence encoding a cytokinin phosphorylase of the invention in antisense orientation, for example StCKPl cDNA in the 3' to 5' direction, and wherein each subsequence is under operable control of different promoters, such as different inducible promotors . Thus, such a nucleotide sequence may differentially express introduced sense and/or antisense sequences, on the application of appropriate different promoter inducing stimuli. The advantage of such a sequence is that tuberisation from the tip of the stolon or shoot or bud or root may be controlled depending on the kind of stimulus applied. Thus, one kind of stimulus may be used to control, typically enhance, the degree of tuberisation and, independently, a second stimulus may be used to control the germination of tubers produced by such a plant. Thus, the eventual sprouting, that is to say, outgrowth of dormant buds on tubers of the tubers at tuber germination may be enhanced for the benefit of a grower seeking to germinate the tubers, or suppressed to improve the storage properties of the tubers by increasing the effective shelf life.

Again, using examples relating to the production of transformed tuberous plants of Solanum tuberosum, Dioscorea

rotundata and Ipomoea batatas, the following nucleic acid sequences are envisaged:

1. The nucleotide sequence of the invention may encode an mRNA which consists--in the 5' to 3' direction--of (i) a first inducible promoter, (ii) at least one cDNA of a cytokinin riboside phosphorylase enzyme, such as StCKPl, in reverse orientation i.e. 3' to 5' orientation, (III) a terminator, (iv) optionally a further promoter, (v) the coding region of an antibiotic resistance marker gene such as the HPT II gene (hygromycin) and (vi) optionally a further stop codon (vii) a second promoter different from the first promoter, and said further promoter (if present) , vii) at least one cDNA of a cytokinin phosphorylase riboside enzyme, such as StCKPl, in sense orientation i.e. 5' to 3' orientation under operable control of the second promoter, and viii) a terminator. When such a sequence is introduced into the cells of Solanum tuberosum, Dioscorea rotundata or Ipomoea batatas plants, the sequence encoding the mRNA is transcribed. The region of the thus transcribed mRNA which encodes the cDNA in sense orientation is translated on application of a given first inducing stimulus, whilst the region of the mRNA which encodes the cDNA in antisense orientation is transcribed but not

translated on the application of a given second stimulus different to the first, and the mRNA region of the antibiotic resistance marker is translated, if present.

By employing such constructs of the invention in thus transformed tuber-producing plants of the invention, differential expression of cDNA sequences of the invention may be employed for both increasing storage time or shelf life and controlling tuber multiplication on the application of different stimuli. Various permutations of the use of constructs of the invention comprising 2 or more sequences in sense and/or antisense orientation may be envisaged by the skilled addressee.

Thus, it is thought that the antisense RNA molecule or short sequences derived from it in planta, in vivo, or in vitro, such as short interfering RNA (siRNA) , form complexes that are capable of interfering with the mRNA of the protein of interest, such as a cytokinin riboside phosphorylase of the invention, e.g. StCPKl. Thus, the synthesis of functional protein (s), such as cytokinin riboside phosphorylase, having enzymic activity in the stolon tip is prevented or substantially inhibited. As a consequence of the interference of the antisense RNA,

enzyme activity of cytokinin riboside phosphorylase (s) involved in tuberisation from the stolon tip, shoot tip, bud or root is decreased, and the number of tubers that eventuate is lower than that typically found when compared to conventional plant varieties of the same species.

In a further aspect of the invention there is provided a nucleotide sequence (nucleotide sequence according to the invention) comprising a transcriptional regulatory sequence, a sequence under the transcriptional control thereof which encodes an RNA which may consist of a plurality of subsequences, characterized in that the RNA subsequences are antisense RNAs to an mRNA of a protein having an enzymic activity in cytokinin riboside phosphorolysis in plant cells of the stolon tip, shoot tip, bud or root.

The nucleotide sequence may encode in antisense orientation an RNA having any number of subsequences which may comprise more than one siRNA sequence; may comprise at least one siRNA sequence and at least one longer RNA sequence; may comprise at least one longer RNA sequence. Preferably, the number of subsequences is up to 6, and more preferably from 1 to 3.

The nucleotide sequence of the invention also includes functional complementary sense polynucleotide sequences of anti-sense sequences of the invention, that when transcribed in plant material, lead to an increase in tuberisation from stolon tips, shoot tips, buds or roots and hence a higher overall number of developing tubers relative to the number found in native plants, for example of known varieties, of the same species. The skilled addressee will also appreciate that the over-expression of such sense sequences for cytokinin riboside phosphorylase proteins, such as StCKPl, capable of being active in stolon tip, shoot tip, bud or root cells of transformed plants of the invention may also elicit the phenomenon of "co- suppression" through siRNA mediated cytokinin riboside phosphorylase gene silencing in the stolon tip, giving rise to storage organs that comprise fewer developing tubers wherein the number of them is reduced when compared with conventional varieties of plants of the same species.

The promoter is a nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription. The cDNA' s encoding a polynucleotide of the invention, such as the

cytokinin riboside phosphorylase, Solanum tuberosum adenosine/cytokinin riboside phosphorylase 1 (StCKPl), contain at least one type of promoter that is operable in a plant cell. The promoter may be an inducible or constitutive promoter when employed in front of sense sequences of the invention. When a promoter is employed to drive transcription from one or more antisense sequences, the promoter is selected from non-constitutive promoters as known in the art. Preferably, in constructs of use in the present invention, an inducible promoter or tissue-specific promoter is employed, such as a stolon cell specific promoter, for example, the promoter from the ^λ gangly' gene (Stgan) of potato described by Trinidade et al . (2002) Gene 303, 77, is operatively linked to a first and/or second nucleic acid sequence or nucleic acid sequence component as herein defined and as provided by the present invention. As discussed, this enables control of expression of the polynucleotide of the invention. The invention also provides plants transformed with polynucleotide sequences or constructs and methods including introduction of such polynucleotide nucleic acid sequences or constructs into a plant cell and/or induction of expression of said first or where appropriate, a second nucleic acid sequence or

construct within a plant cell, e.g. by application of a suitable stimulus, such as an effective exogenous inducer.

The term "inducible" as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on" or increased in response to an applied stimulus (which may be generated within a cell or provided exogenously) . The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. The preferable situation is where the level of expression increases upon application of the relevant stimulus by an amount effective to alter a phenotypic characteristic. Thus an inducible (or

"switchable") promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about a desired phenotype (and may in fact be zero) . Upon application of the stimulus,

expression is increased (or switched on) to a level, which brings about the desired phenotype. One example of an inducible promoter is the ethanol inducible gene switch disclosed in Caddick et al (1998) Nature Biotechnology 16: 177-180.

Chemically regulated promoters can be used to modulate the expression of a gene or a polynucleotide sequence of the invention in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemically inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemically inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid. Other chemically regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et

al. (1998) Plant J. 14 (2) : 247-257) and tetracycline- inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) MoI. Gen. Genet. 227:229-231 , and U.S. Patent Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Where enhanced expression of a cytokinin riboside phosphorylase of the invention, or a homologue thereof, for example, for the generation of increased numbers of tubers, the DNA may be placed in a construct of choice which comprises a cytokinin riboside phosphorylase nucleic acid sequence, such as StCKPl, or a homologue or orthologue thereof, in the sense orientation, that is to say, the 5' to 3' direction, for the generation of mRNA from which a cytokinin riboside phosphorylase or a functional fragment thereof for use in the invention may be translated. It has been found that enhanced expression of StCKPl gives rise to an increased incidence of stolon tip enlargement which in turn gives rise to the generation of increased numbers of tubers. Furthermore, enhanced expression of StCKPl in the tuber has been found to prolong endodormancy and that this can be reversed after the application of a physical stimulus, such as chilling.

Use of the terms "homology" and "homologous" and "homologue" herein does not imply any necessary evolutionary relationship between compared sequences, in keeping for example with standard use of terms such as "homologous recombination" which merely requires that two nucleotide sequences are sufficiently similar to recombine under the appropriate conditions. Further discussion of polypeptides according to the present invention, which may be encoded by nucleic acid according to the present invention, is found below.

The present invention also encompasses plant cells, for example, stolon tip plant cells comprising the polypeptide expression product of a nucleic acid molecule encoding a cytokinin riboside phosphorylase or a functional fragment thereof according to the invention as disclosed herein or obtainable in accordance with the information and suggestions herein. Also provided are methods of making such an expression product by expression from a nucleotide sequence encoding for it under suitable conditions in suitable host cells e.g. E.coli. Those skilled in the art are well able to construct vectors and design protocols and systems for expression and recovery of products of recombinant gene expression.

The cytokinin phosphorylase of the invention is contemplated to be any protein or polypeptide that displays cytokinin riboside phosphorylase activity, such as StCKPl, and is capable of effecting tuberisation in a stolon tip plant cell, plant tissue or stolon-producing plant into which it is introduced and that may be produced by the method of the invention. Thus, a polypeptide according to the present invention may be an allele, variant, fragment, derivative, mutant or homologue of the (a) polypeptides as mentioned herein. The allele, variant, fragment, derivative, mutant or homologue may have substantially the same function of the polypeptides alluded to above and as shown herein or may be a functional mutant thereof.

"Homology" in relation to an amino acid sequence or polypeptide sequence produced by the method of the invention may be used to refer to identity or similarity, preferably identity. As noted already above, high level of amino acid identity may be limited to functionally significant domains or regions.

In certain embodiments, an allele, variant, derivative, mutant derivative, mutant or homologue of the

specific cytokinin riboside phosphorylase sequence may show little overall homology, say about 20%, or about 25%, or about 30%, or about 35%, or about 40% or about 45%, with the specific sequence. However, in functionally significant domains or regions, the amino acid homology may be much higher. Putative functionally significant domains or regions can be identified using processes of bioinformatics, including comparison of the sequences of homologues .

Functionally significant domains or regions of different polypeptides may be combined for expression from encoding nucleic acid as a fusion protein. For example, particularly advantageous or desirable properties of different homologues may be combined in a hybrid protein, such that the resultant expression product, may include fragments of various parent proteins, if appropriate.

Similarity of amino acid sequences may be as defined and determined by the TBLASTN program, of Altschul et al. (1990) J. MoI. Biol. 215: 403-10, which is in standard use in the art. In particular, TBLASTN 2.0 may be used with Matrix BLOSUM62 and GAP penalties: existence: 11, extension: 1. Another standard program that may be used is

BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711). BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Adv. Appl. Math. (1981) 2: 482-489). Other algorithms include GAP, which uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. As with any algorithm, generally the default parameters are used, which for GAP are a gap creation penalty = 12 and gap extension penalty = 4. Alternatively, a gap creation penalty of 3 and gap extension penalty of 0.1 may be used. The algorithm FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448) is a further alternative .

Where expression in particular tissues is desired, tissue- specific promoters can be utilized, for example, in the stolon using the promoter from the ^λ gangly' gene (Stgan) of potato (Trinidade et al supra) . Other tissue-specific promoters include the tuber-specific promoter B33 described

by Rocha-Sosa et al (1989) EMBO J. 8, 23-29; Zourelidou et al. (2002) Plant J. 30(4), 489-497, and the tuber cortex specific promoter described by Korobczak et al . (2005) Plant Science 168, 339 et seq.

So-called constitutive promoters that drive transcription of nucleic acids of the invention in the sense orientation i.e. 5' to 3' direction may also be used in the methods of the present invention. Constitutive promoters include, for example, CaMV 35S promoter (Odell et al. (1985) Nature

313:810-812) ; rice actin (McElroy et al. (1990) Plant Cell 2:163-171) ; ubiquitin (Christensen et al . (1989) Plant MoI. Biol. 12:619-632 and Christensen et al . (1992) Plant MoI. Biol. 18:615-689) ; pEMU (Last et al . (1991) Theor. Appl . Genet. 81:581-588); MAS (Velten et al . (1984) EMBO J. 3:2723-2730) ; ALS promoter (U.S. Application Serial No. 08/409,297), and the like. Other constitutive promoters include those in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

Examples of promoters suitable for use in DNA constructs of the present invention include viral, fungal, bacterial, animal and plant derived promoters capable of functioning

in plant cells. It will be appreciated that the promoter employed should give rise to the transcription of a sufficient amount of the antisense RNA molecule, that is to say, the cytokinin riboside phosphorylase antisense RNA molecule, at a rate sufficient to cause an lowering of cytokinin riboside phosphorylase activity in plant cells of, for example, the stolon tip, shoot tip, bud or root. The required amount of antisense RNA to be transcribed may vary from plant to plant. Generally, in plants and plant cells of the invention inducible or developmentally regulated promoters that are capable of being used in stolon structures, such as plant cells of the stolon tip, shoot tip, bud or root are preferred.

Naturally, the man skilled in the art will appreciate that terminator DNA sequences will be present in constructs used in the invention. A terminator is contemplated as a DNA sequence at the end of a transcriptional unit which signals termination of transcription. These elements are 3 ' -non- translated sequences containing polyadenylation signals which act to cause the addition of polyadenylate sequences to the 3' end of primary transcripts. Examples of terminators particularly suitable for use in nucleotide sequences and DNA constructs of the invention include the

nopaline synthase polyadenylation signal of Agrobacterium tumefaciens (A. Depicker et al . , 1982, J. of MoI. & Applied Gen. 1:561-573), the 35S polyadenylation signal of CaMV, octopine synthase polyadenylation signal and the zein polyadenylation signal from Zea mays. Sequences mentioned above may be isolated from fungi, bacteria, animals or plants .

The invention also provides a DNA construct comprising a nucleotide sequence according to the invention (in either sense or antisense configuration) , as well as a biological vector comprising the said sequence or construct. The biological vector may be a virus or bacterium, such as Agrobacterium tumefaciens, for example, and the construct advantageously further encodes a marker protein, such as one having herbicide resistance, or anti-bacterial properties .

DNA constructs and nucleotide sequences of the invention may be used to transform cells of tuber-producing plants that produce tubers in various ways known in the art. Plants transformed with DNA segments containing sequences of interest as provided herein may be produced by standard techniques, which are already known for the genetic

manipulation of plants. DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti- plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-O 116718, NAR 12(22) 8711 -87215 1984), particle or micro projectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al . (1987) Plant Tissue and Cell Culture, Academic Press), electroporation (EP 290395, WO 8706614) other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611), liposome mediated DNA uptake (e.g. Freeman et al. Plant Cell Physiol. 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U.S.A. 87: 1228 (199Od) Physical methods for the transformation of plant cells are reviewed in Oard, 1991, Biotech . Adv. 9: 1-11. Thus once a nucleic acid sequence or gene has been identified, it may be reintroduced into plant cells using techniques well known to those skilled in the art to produce transgenic plants of the appropriate phenotype. Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species.

Micro projectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient

or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, e.g. bombardment with Agrobacterium coated micro particles (EP-A-486234) or micro projectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233) .

Following transformation, a plant may be regenerated, e.g. from single cells, callus tissue, pro-embryogenic masses or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al . , Cell Culture and Somatic Cell Genetics of Plants, Vol. I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weiss Bach and

Weiss Bach, Methods for Plant Molecular Biology, Academic Press, 1989.

The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation

system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.

Known methods for transforming tuber-producing crop plants of the invention include, for example, those taught by Tor M et al Plant Cell Reports Vol. 12, Numbers 7-8, May 1993 pp 462-467 (Dioscorea rotundata) ; Moran R Plant Science, vol. 139, No.2, 31 December 1998 pp 175-184 (Ipomoea batatas) , and in many cases transformed plant cells may be cultured to regenerate whole plants which can subsequently reproduce to give successive generations of genetically modified plants.

The invention still further provides eukaryotic cells, such as plant cells (including protoplasts) for example, containing a nucleotide sequence, a DNA construct or vector of the invention.

The invention still further provides transformed tuber- producing plants of the invention comprising transformed stolon tip, shoot tip, bud or root cells, the progeny of such plants which contain the sequence stably incorporated and hereditable in a Mendelian manner, and/or the seeds of

such plants or such progeny. Such plants include field crop plants and ornamentals including varieties of Solanum tuberosum (potato) , Dioscorea spp (yams) , Ipomoea batatas (sweet potato) , Helianthus tuberosum (Jerusalem artichoke) , Manihot esculenta (cassava, manioc) , Pachyrhizus erosus (jicama), Xanthosoma spp (malanga) , Colocasia esculenta (taro) , Eleocharis dulcis (water chestnuts) , Oxalis tuberosa (oca) , Maranta arundinacea (arrowroot) , Allium oschaninii (shallot), A. sativum (garlic), and A. schoenoprasum (chives) . Certain ornamental plants may also produce tubers as defined herein, such as varieties of tulip, amaryllis, hyacinth, daffodil, narcissus, cyclamen, lily, lily of the valley, iris, gladiolus, crocus, crocosmia, snow drop, bluebell, dahlia e.g. varieties ^λ Fuzzy Wuzzy' and ^λ Boogie Woogie' (online Sutton Seeds catalogue 2007), freesia, gloxinia, anemone, fritillaria, alstromeria ligtu and hybrids thereof, camassia esculenta, arum italicum, muscari, agapanthus, begonia, acidanthera, ranunculus, ornamental allium and the like. Plants and/or plant cells of Solanum tuberosum, Dioscorea rotundata and Ipomoea batatas are particularly preferred.

The invention still further provides the use of a sequence according to the invention, whether "naked" or present in a

DNA construct or biological vector in the production of eukaryotic cells, particularly plants comprising stolon tip cells having modified stolon tuberisation patterns of activity when compared with that of conventionally produced plants of the same species or of varieties thereof.

The invention still further provides a method of inducing an under expression of a cytokinin riboside phosphorylase in plant cells comprising introducing into such cells a nucleotide sequence according to the invention, or a construct or vector containing it.

In a further aspect of the invention there is provided use of at least one nucleic acid sequence in sense orientation of a cytokinin riboside phosphorylase of the invention, such as StCKPl, in increasing the mean number of tubers produced by transformed tuber-producing plants of the invention when compared with the mean number of tubers found in non-transformed plants of the same species. Such a use is commercially valuable for producing further tubers for commercial multiplication and "seed" tuber sales and/or for food sales-related purposes.

In a further aspect of the invention there is provided use

of at least one nucleic acid sequence in antisense orientation of a cytokinin riboside phosphorylase, such as StCKPl, to alter the mean duration of endodormancy, for example, by shortening the mean duration of endodormancy of tubers produced from stolons, shoots, buds or roots in transformed tuber-producing plants of the invention relative to the mean duration of endodormancy of tubers found in non-transformed plants of the same species. λEndodormancy' refers to a dormant state in which the plant, or some part of the plant, maintains itself in a state of suspended animation with substantially no growth and minimal metabolism even though conditions are ideal for normal metabolism and growth. At some point in the winter, over-wintering native tubers and seeds shift spontaneously from the endodormant state to a state of ^λ imposed dormancy' in which only the low temperature (ie environmental constraint) prevents them from growing. Such a use is commercially valuable for the storage of tubers in native tuber-producing plants of the invention. However, in the case of tubers harbouring antisense constructs of the invention, endodormant states can be shortened thus permitting the grower for the first time to synchronise sprouting from tubers. As a further aspect of the invention, there is provided use of a cytokinin riboside

phosphorylase nucleotide sequence (s) in antisense orientation in transformed cells of a tuber for synchronising sprouting from a tuber storage organ. By employing tubers comprising tuberous cells transformed with antisense sequence (s) of a cytokinin riboside phosphorylase nucleotide sequence, such as an antisense sequence of StCKPl, the grower is able to synchronise crop growth and tuber production more efficiently. At the domestic scale, tubers comprising antisense sequences of a cytokinin riboside phosphorylase of the invention would not require chitting, that is to say, the provision of a stimulus to promote sprouting, and as a consequence the growing of a synchronised crop of tuber-producing plants becomes more facile. As a still further aspect of the invention, there is provided use of at least one nucleic acid sequence in antisense orientation of a cytokinin riboside phosphorylase in shortening the mean duration of endodormancy of tubers produced by a transformed tuber-producing plant relative to the mean duration of dormancy in tubers of non-transformed plants of the same species. Tubers comprising such antisense sequences may have their mean duration of dormancy shortened. It is therefore possible to provide for more than one crop per annum in crop plants of the invention that typically comprise antisense sequences of

the invention.

The teaching of all references cited herein is incorporated in its entirety into the present description.

Detailed Description of the Invention

There now follow non-limiting examples and figures illustrating the invention.

FIGURE LEGENDS

Figure 1. Cytokinin-binding activity becomes detectable prior to visible swelling of stolon tips .

a, Structures of cytokinins. dHZ, dihydrozeatin; iP, isopentenyladenine; iPR, iP riboside; iPRDP, IPR 5'- diphosphate; iPRMP, iPR 5 ' -monophosphate; Z, zeatin; ZR, Z riboside. b, Potato tubers kept in paper sacks at 15°C for six months after lifting, i.e. in April, develop clusters of stolons but no tubers: ^λ pre-tuberising' stage (upper). By nine months, i.e. in June, there is a mixture of swollen ^λ tuberising' and not-yet-swollen ^λ incipiently tuberising' stolons (lower) . c, Amount of a labelled form of Z (ZR-

[ ³ H] diol) bound plotted against concentration of crude soluble protein (50 to 75% saturated (NH ₄ ) ₂ SO ₄ cut) from potato tissues. Binding activity relative to binding by bovine serum albumin (BSA) was detected in incipiently tuberising stolon tips (June), i.e. prior to swelling, but not in pre-tuberising tips 3 months earlier (April) or other tissues, . Values are the mean of two experiments, deviations between 3 & 6%. Scale bar, 5mm (b) .

Figure 2. StCKP1-related transcripts are most abundant in the shoot apex, and become more abundant in stolon tips on tuberisation.

a, Northern blot of RNA from potato tissues probed with a 437 bp fragment of StCKPl. SA, shoot apex; L, leaf; ST, stem; F, flowers; R, roots; S, sepals; P, petals; C, carpels; SN, stamens; T, tuber. b. RT-PCR estimation of StCKPl abundance relative to potato tubulin (StTUBl) in stolon tips at successive stages. Agarose gel electrophoretogram (upper) yielding digitized data for the chart of StCKPl relative abundance (lower) . PT, pre- tuberising; IT, incipient tuberising; T, tuberising.

Figure 3. StCKPl encodes a cytokinin riboside phosphorylase .

a, StCKPl amino acid sequence of nucleosidase family conserved motif II. h, bulky aliphatic residue; s, small; &, bulky hydrophobic; •, any residue, E, predicted β sheet; C, predicted random coil. Residues matching the consensus on black, non-matches on red. Critical alanine residue asterisked. b, The enzyme reaction. c, HPLC analysis of the products from 50μM cytokinin and ImM inorganic phosphate (Pi) or ribose 1-phosphate (RlP) . Each trace shows the peaks of UV absorbance (y axis) due to cytokinins eluting at times (x axis) corresponding to the R _τ of standard cmpds (Std) .

Figure 4. StCKPl expression is necessary for tuberisation in vitro.

a, single node explants of potato shoot cultures after 28d on tuber induction medium with 5μM β-estradiol, the XVE inducer. Axillary buds of empty vector control lines produce a terminal tuber on a short stolon (upper two lines), XVE:: StCKPl antisense lines grow a stolon with no terminal tuber (lower three lines) , occasionally a small

lateral tuber (b) . (c) , Map of iP synthesis with location of CKP. 1, IPT; 2, nucleotidase; 3, nucleosidase; 4, LOG; 5, CKP. d, Proposal for a switch in cytokinin source at the transition into tuberisation . Once arrested, the inactive stolon apical meristem will not synthesise cytokinin and CKP maintains the high sink strength of the stolon tip for the growing tuber by releasing hormone from transported riboside. Scale bars, 5mm (a and b) ; and 5mm (d) .

Figure 5. Scatchard analysis of cytokinin-binding by stolon tip soluble protein

ZR- [ ³ H] diol binding data for soluble protein (50-75%

(NH ₄ ) ₂ SO ₄ ^cuf ) from potato stolon tips at incipient tuberisation. Each point is the mean of results from duplicate experiments; deviations from the mean were 9 to 16%.

Figure 6. SDS-PAGE separation of affinity-prepared potato stolon tip cytokinin binding proteins .

Lane 1, CKBP purified using affinity chromatography from potato stolon tips at incipient tuberisation. Lane M, SDS-7 molecular weight markers. Sizes in kDa are at right.

Figure 7. Amino acid sequence predicted from StCKPl ORF .

Figure 8. Genomic organisation of potato StCKPl gene(s).

Genomic DNA from potato cv Majestic digested with: Hindi I I (H), BamRI (B), Xhol (X), EcoRI (E), Hindi (Hc) and Mspl (Ms) . A plasmid containing the ORF of StCKPl was digested with Xhol and included on the gel as a control (Dp) .

Fragments were hybridised with the ORF of StCKPl. Marker (M) sizes at right.

Figure 9. Alignment of translated ESTs that contain the 19 amino acid sequence.

The region highlighted in blue is the original amino acid sequence obtained from the NH ₂ -terminal sequencing. Le - EST from Lycopersicon esculentum flowers (gil6239544) , Str -

EST isolated from Solanum tuberosum (St) roots (gil7075814) , Sts - EST isolated from St swollen stolons (gi42511149) , StI - EST isolated from St leaves (gil2588117) , Stst - EST isolated from St stolon (gi9562478), Stm - EST isolated from St mixed tissue (gi21916199) , Sta - EST from St plants subjected to abiotic stress (gi39829888) . Numbers on the right refer to amino acid position from the N-terminal methionine, if present.

Multiple sequence alignment using ClustalW (Chenna et al . ,

2003) * identical amino acids, ^λ :' conserved amino acids, ^λ . ' semi-conserved amino acids

Figure 10a Nucleotide sequence of StCKPl cDNA. Nucleotides are numbered from the start of the cDNA sequence and The gap between the 54 ^th and 55 ^th nucleotide in the sequence denotes the first in-frame ATG. ^ - signal cleavage site (PSORT, Nakai & Horton, 1999); dashed line, potential polyadenylation signal.

Figure 10b Amino acid sequence of StCKPl

The amino acid sequence was translated starting with the first in-frame ATG and amino acids are numbered from the initial methionine.

Figure 11 Nucleotide sequences derived from the StCKPl cDNA.

The open reading frame (ORF) includes the stop sequence.

Figure 11a. Full length ORF from cDNA, sense strand (inclusive of signal peptide sequence)

Figure lib. Full length ORF from cDNA, inverse complement

(inclusive of signal peptide sequence)

Figure lie. ORF from cDNA, sense strand minus signal peptide sequence

Figure Hd. ORF from cDNA, inverse complement minus signal peptide sequence

Figure 12 Duration of (a) chill-sensitive and (b) chill- insensitive dormancy in potato tubers in which the expression of StCKPl has been partially suppressed by virus-induced gene silencing

EXAMPLES SECTION

It is thought that factors allowing a subset of cells to respond to cytokinin will be proteins that interact with the free base or its immediate precursors: cytokinin ribosides or ribotides. The inventors detected such proteins by cytokinin-binding, selecting as source material tissue undergoing a cytokinin-mediated process at a predictable time: viz. the tips of stolons growing on tubers in darkness, just before they spontaneously tuberise. This experimental system has no treatment artifacts because the change in development takes place in constant conditions in the intact plant under the control of endogenous changes. Also, shoot apices, the target tissue for most cytokinin responses, are accessible and so can be harvested without dissection trauma artifacts.

The source material for analysis was generated by incubating potato tubers (cv ^λ Majestic') long term in paper sacks at 15°C. The first sprouts appeared in mid-December and by April, the sacks were filled with branching stolons with no sign of the proximal swelling at the tips indicative of secondary tuberisation . Stolon tips were harvested at this stage as ^λ pre-tuberising' (PT) tissue. In June, the first secondary tubers appeared, followed within days by widespread tuberisation. At this stage, ^λ incipiently tuberising' (IT) tissue was collected. This comprised still untuberised tips from stolons also carrying some tuberising tips (Fig. Ib). Incipiently tuberising tissue is visually indistinguishable from the pre- tuberising tissue collected in April.

Protein from stolon tips was assayed for cytokinin binding by equilibrium dialysis against a tritiated derivative of zeatin riboside (ZR- [ ³ H] diol) ¹³ at 5 nM. Binding to membrane fractions was not detected (data not shown) . Soluble protein from stolon tips at incipient tuberisation bound approximately 8 times more cytokinin per mg of protein than that from pre-tuberising stolons (Fig. Ic) . Binding activity in the pre-tuberising stolons was of the same order as that for bovine serum albumin (BSA) at the same

total protein concentration, i.e. there was a spontaneous increase in binding activity in the stolon tip before spontaneous tuberisation .

Tissue specificity was investigated by assaying cytokinin binding in protein from leaves, mature potato tubers and microtubers (tubers induced to grow on stolon tips in vitro). For all of these sources, levels of ZR- [ ³ H] diol binding activity were not detectably greater than that of BSA (Figure Ic), i.e. binding activity is a specialized, not a general, feature.

Binding specificity was investigated by competition between ZR- [ ³ H] diol and 2000-fold excesses of various unlabelled compounds (Table 1). Excess ZR-diol reduced binding activity to a level comparable to non-specific binding. Unlabelled zeatin riboside and zeatin were highly effective at competing with ZR- [ ³ H] diol. The non-cytokinin structural homologues adenine and adenosine inhibited the binding of ZR- [ ³ H] diol but by less than excess cytokinins. Therefore binding was saturable, with some specificity for cytokinins. Incipiently tuberising stolon tip protein appeared to bind the less active ¹⁴ cytokinin metabolite, dihydrozeatin riboside (Fig Ia) , almost as effectively as

zeatin, indicating that binding is due to proteins other than action receptors.

The affinity of the protein for ZR- [ ³ H] diol was estimated by Scatchard analysis (Figure 5) . The cytokinin-binding site concentration in the extract was calculated to be 30 nM, with a dissociation constant (K _d ) of 1.7 x 10 ^~7 M for ZR-

[ ³ H] diol and an estimated binding site concentration of ~70 pmol q ^'1 FM stolon tips.

A cytokinin-binding protein (CKBP) fraction was isolated from the soluble protein of incipiently tuberising potato stolon tips by affinity chromatography on avidin-Sepharose pre-loaded with biotinylated zeatin riboside. Soluble protein from 40 g stolon tips was cycled through an avidin column to remove biotinylated proteins. The protein extract minus biotinylated proteins was then applied to the cytokinin pre-loaded column and cycled 6 times through the column. Unbound proteins were washed off before CKBPs were eluted in an excess of biotin. The fractions corresponding to a peak of UV absorbance were concentrated using molecular weight filters. The concentrated CKBP protein fraction was analysed by SDS-PAGE (Figure 6) . The most prominent band of protein had an estimated M _r of 40 kDa .

This band was electroblotted and a 19 amino acid NH ₂ - terminal sequence obtained. Other components of the affinity-isolated CKBP fraction are under investigation.

The NCBI EST database was searched for homologues using the translated BLAST search programme. 100% identity was found between the sequence and translated EST sequences from potato, tomato and tobacco from a variety of tissue types including: roots, flower buds, germinating seedlings, shoot meristems, stolons, sprouting eyes of potato tubers and microtubers. Primers: Forward: AGC AAA TGG TGC TAT TAG TGG AAA GAC and Reverse: CTT GTG TAA TCT CCA TTC ACT TCA AGC, designed to consensus regions of the ESTs and used to amplify a partial clone out of cDNA from incipiently tuberising stolon tips, which was then cloned and sequenced. The annealing temperature used was 55°C.

RACE-PCR was used to amplify the open reading frame (ORF) and 5' and 3' UTRs. A potential long ORF from the nucleotide in position 54 to a termination codon at position 1116 was identified (Figure 7) encoding a polypeptide of 354 residues with a calculated molecular weight of 39260. The translation product is predicted by PSORT to have a signal peptide of 14 residues likely to be

removed during processing ¹⁵ , leaving a mature polypeptide consisting of 340 residues with a calculated molecular weight of 37785. Southern analysis (Figure 8) suggests the presence of multiple copies of related genes in potato - a tetraploid plant.

Steady-state levels of expression were investigated by Northern analysis. The RNA sequence was only abundant in the shoot apex, with small amounts in the leaf, root and carpel (Fig. 2a). RT-PCR showed transcripts apparently more abundant in tuberising stolons than in incipiently tuberising and pre-tuberising stolons (Fig. 2b).

The full length sequence was used to search the NCBI database using pBLAST. Computation was performed at the Swiss Institute of Bioinformatics using the BLAST network service. The program was BLASTp, the database was UniProt (SwissProt/TrEMBL) , and the matrix 'blosum 62'. Gapped alignment, BLAST filter and Xblast-repsim filter were all 'on', and Coils filter 'off. Open gap penalty, extend gap penalty and drop off value were all set to 'default'. Mismatch penalty was set to -3 and match reward to +1.

The only similar proteins characterized to date are a group classed as vegetative storage proteins of poplar, some of which are wound-inducible ¹⁶ . The sequence was also found to share similarity in three distinct, conserved motifs (I, II and III) with members of a class of largely bacterial nucleosidases, described by Mushegian & Koonin ¹⁷ , who predicted that the probability of obtaining each of the conserved motifs by chance alone was less than 10 ^~5 . The predicted amino acid sequence of the potato protein differs from the consensus sequence for both motifs I and II at two positions (highlighted in red in Figure 3a) , but matches the consensus sequence for motif III. The functions of motifs I and III are unknown, but conserved motif II shows low similarity to the ribose-binding motif of eukaryotic nucleoside phosphorylases . In particular, the potato protein motif II includes a conserved alanine between two β-sheets (Fig 3a) , a central feature of the active centre of a human purine nucleoside phosphorylase where it forms a hydrogen bond with the ribosyl 3 ' -hydroxyl ¹⁸ .

The ORF was expressed in E.coli fused to a maltose-binding protein and the protein shown to have cytokinin riboside phosphorylase activity (Fig. 3b). The purified protein converted 2μM isopentenyladenine entirely to

isopentenyladenosine in the presence of ribose 1-phosphate, and 2μM isopentenyladenosine to isopentenyladenine in the presence of phosphate (Fig. 3c). Comparable conversions were obtained with zeatin or dihydrozeatin and ribose 1- phosphate, and with zeatin riboside or dihydrozeatin riboside and phosphate (data not shown) . In the absence of inorganic phosphate, the ribosides remained unaltered (Fig. 3c) , showing that the protein has no detectable cytokinin riboside hydrolase activity. Using ELISA ¹⁹ to quantitate activity, the K _m for isopentenyladenine, zeatin and dihydrozeatin was approx. 4μM, and isopentenyladenosine was an inhibitor, eliminating activity at 250μM and above. A spectrophotometric assay ²⁰ showed that the enzyme also catalyses the adenosine phosphorylase reaction in both directions, with a K _m for adenine of 34μM, and complete inhibition by adenosine above 250μM. These characteristics resemble those reported for wheat germ adenosine phosphorylase ²¹ .

The estrogen-inducible promoter XVE ²² was used in antisense plants. Shoot cultures of inducible antisense lines were not visibly different from empty vector transformants or wild type, even after estradiol treatment. However, a marked difference from controls was revealed when single

node explants were tested for induction of a microtuber from the axillary bud in response to cytokinin. On medium containing estradiol and cytokinin, explants from empty vector controls produced a terminal tuber, either directly in the axil or at the end of a short stolon. For explants from all seven transgenic lines (independent transformation events) expressing the antisense version of the gene, the axillary bud grew out as a long thin stolon and had no tuber at the apex (Figure 4a: in some cases, a single small tuber formed in the axil of a scale leaf on the stolon - example at 4b - axillary buds on the stolon are weaker phloem sinks than the stolon apex and so don't always receive enough estradiol to induce the antisense transgene) . We conclude that the cytokinin phosphorylase is necessary for the transformation of stolon tips into developing tubers.

This transformation is promoted by cytokinin ⁴ ' ⁵ . Moreover, when the cytokinin synthesis gene ipt was expressed in the leaf primordia and apical meristem periphery of axillary buds of tobacco, tubers formed ²³ , i.e. a local cytokinin increase can force stem tuber development in non-tuberous plants. It follows that, for the initiation of potato tubers, cytokinin phosphorylase is catalysing

phosphorolysis of cytokinin ribosides to increase the availability of the active free base, as expected for the large excess of inorganic phosphate over ribose 1-phosphate in living tissues ²⁴ . We note that the growth of stolons was not suppressed as a result of antisense suppression of the enzyme, indicating that this protein is not necessary for stolon apical meristem activity.

In summary, potato stolon tips exhibiting increased cytokinin-binding activity at the point of tuberisation, a cytokinin-promoted event, were the source material for affinity chromatography on a cytokinin column. The principal protein isolated is shown to interconvert highly active cytokinin bases and the less active transport form, cytokinin ribosides. Suppressing the expression of the protein blocked tuberisation, but not growth, at the stolon tip .

From the enzymic activity of the protein it encodes, we designate the novel gene ^x S_olanum tuberosum Adenosine/ £ytok.inin riboside phosphorylase JL' (StCKPl) .

We propose that the cytokinin riboside phosphorylase activity of StCKPl protein is an alternative route for the

generation of active cytokinin bases (Fig 4b) which uses cytokinin ribosides arriving from other parts of the plant. For potato stolons, transport to the tip occurs in the phloem and off-loading is apoplastic, but after tubers are initiated, unloading changes to symplastic ²⁵ . LOG, the cytosolic phosphoribohydrolase generating free base for the apical meristem, uses cytokinin nucleotides as substrate ⁷ . Cytokinin nucleotides may be synthesized in the active meristem or generated from ribosides transported in from other parts of the plant, so the two enzymes will compete indirectly for incoming cytokinin ribosides. The outcome of that competition will influence whether a potato stolon tip begins to tuberise or continues to grow as a stolon; a local increase in the phosphorylase is likely to tip the balance, capturing for tuberisation cytokinin that might otherwise go on to sustain the apical meristem (Fig. 4c) .

There is only one record of cytokinin riboside phosphorylase activity in plants, reported by Chen and Petschow in 1978 ²¹ . All other recorded attempts to detect cytokinin nucleoside phosphorylase in plants failed ²⁶ ' ²⁷ , and the activity is no longer included in metabolic maps of cytokinin interconversion ⁷ . Our results are a re-discovery of the enzyme, restoring it to the map (Fig. 4b) . The

enzyme is not restricted to stolon tips at tuberisation as evidenced by the wheat germ study ²¹ , and suggested by the sequences of wound-induced and bark proteins ¹⁶ , and the results of Northern analysis (Fig. 2a). Interestingly, wheatgerm, bark and tubers are storage tissues, and cytokinins are hormones that enable tissues to be nutrient sinks ²³ .

METHODS

Cytokinin-binding activity

Methods described in Supplementary Information

Sequence analysis

For Southern analysis DNA was extracted from potato tissue ²⁸ and hybridised overnight to the StCKPl ORF labelled with [α- ³² P]dCTP by random priming. For Northern analysis total RNA was extracted from potato tissue using the RNeasy Plant Mini Kit (Qiagen) and probes labelled by random priming.

Protein expression

For maltose binding protein tagged recombinant proteins, the ORF of StCKPl was ligated into pMAL (C2X) (NEB) and expressed in Rosetta 2 (DE3)pLysS E.coli strain (Novagen) . The enzyme reaction was determined by incubating the purified recombinant enzyme with 50μM substrate at 3O ⁰ C for

10 minutes ²⁹ and identifying the products by HPLC. Adenosine phosphorylase was assayed spectrophotometrically ²⁰ and cytokinin riboside phosphorylase by ELISA ¹⁹ of the reactants. K _m s were determined at pH 7.2.

SUPPLEMENTARY METHODS

Preparation of soluble protein from stolon tips

Solanum tuberosum L. cv Majestic tubers grown in Cambridge

Botanic Garden were kept at 15°C in paper sacks. Stolon tips, 5 to 10mm, were harvested at 6 months (pre- tuberising, PT) and 9 months (diam. equal to that of PT = 'incipient tuberising' , IT; diam. >2xPT = ^λ tuberising' , T), snap frozen in liq. N ₂ and stored at -70 ⁰ C before grinding in liq. N ₂ with 0. Ig g g ^"1 FM PVPP, extraction in 3 vol EM

(ImM PMSF, 4mM glutathione, 0. IM sodium ascorbate ImM

EDTA. Na ₂ , 5mM MgCl ₂ , 5OmM Tris-HCl, pH 7.4), and

centrifugation (100Og for 20min, then 3200Og for Ih) . The 50 to 75% (NH ₄ ) ₂ SO ₄ cut was isolated from supernatant, and G-25 de-salted into EM without ascorbate.

Isolation of cytokinin-binding proteins by affinity chromatography

Biotinylated zeatin riboside was synthesised by coupling 3μmol of periodate-oxidised zeatin riboside and 5μmol biotin long chain hydrazide (Vector) , stabilising the linkage with borohydride, and HPLC-purifying the product. Naturally biotinylated proteins were removed from the stolon tip soluble proteins by passage through unmodified monovalent avidin sepharose (Pierce) at 4°C before the cytokinin-binding fraction was isolated on an avidin sepharose column pre-charged with biotinylated ZR, eluted in 2mM biotin, ImM EDTA, Na ₂ , 5mM MgCl ₂ , 5OmM Tris-HCl, pH 7.4, and concentrated using 1OkDa MW filters (Nanosep) .

Assay of cytokinin-binding activity by equilibrium dialysis

A radiolabelled derivative (ZR- [ ³ H] diol, 150GBq mmol ^x ) was synthesised by reducing periodate-oxidised zeatin riboside with [ ³ H]NaBH ₄ and HPLC-purifying the product ¹³ . Protein

samples were dialysed for 14h at 4°C against 5 nM ZR- [ ³ H] diol, ImM EDTA, Na ₂ , 5mM MgCl ₂ , 5OmM Tris-HCl, pH 7.4 in ImI cells separated by 5000 MW cut-off membrane in a Dianorm-Gerate dialyser, before liquid scintillation analysis.

RT-PCR cDNA was transcribed from 2μg RNA and multiplex PCR carried out with primers for a 265bp region of StCKPl and 450bp region of StTUBl. Products were resolved by agarose gel electrophoresis and fluorescence data from the ethidium bromide-stained gel used to calculate the ratio of StCKPl to StTUBl. Primer sequences, StCKPl ₁ F: GCT TCT CTC AAA CTC AAA GTG TCT ACC, R: GAG ATC ACC AGG TTT GTA ACT AGG ATG; StTUBl, F: AGT GCC AGA GCT TAC TCA AC, R: CAC CTG TCT ACC AAT GCA AG.

TABLES

Table 1. ZR- [ ³ H] diol binding by soluble potato protein from incipiently tuberising stolon tips in the presence of 2000- fold excess of unlabelled compounds (values are means of duplicate experiments, duplicates varying by 3 to 6%) .

The novel StCKPl gene regulates the duration of tuber dormancy

Summary

The aim of this work was to test for regulatory effects of the novel gene on the duration of potato tuber dormancy. Our physiological work on the control of the end of dormancy by cytokinins and our biochemical work on the interconversion of cytokinins by the StCKPl protein predict that the gene will affect the duration of dormancy, or the dormancy-breaking effect of chill-treatment, or both. We used virus-induced gene silencing to generate potato tubers in which expression of the novel gene had been partially suppressed to different degrees, and determined both the extent to which the gene had been down-regulated, and the mean duration of dormancy in chill-treated and non-chilled tubers, for each infected plant. The duration of dormancy for un-chilled tubers minus the duration of dormancy for chilled tubers is the extent of the response to the chill treatment - this is the ^λ chill-sensitive' component of dormancy. The duration of dormancy for chilled tubers is

the ^λ chill-insensitive' component of dormancy. These parameters are plotted in Fig 12 which shows that changes in the expression of StCKPl had no apparent effect on the duration of the chill-insensitive component of dormancy, but the mean duration of chill-sensitive dormancy was directly proportional to the abundance of transcripts from the gene. We reason that by increasing the expression of StCKPl in tubers, or selecting for variants with increased expression of StCKPl, we will generate improved lines with prolonged endodormancy that is susceptible to reversal following chill treatment by the grower wishing to obtain tuber germination.

Technical details

PVX. CKP, the agent for gene silencing, was generated by cloning in antisense orientation a 437bp fragment from StCKPl cDNA into the PVX vector pGR106. This fragment was selected as common to all the variant forms of StCKPl identified as ESTs in the databases, with the aim of silencing as many of the similar genes as possible. The construct PVX. PDS contains a 412 bp cDNA fragment of a potato phytoene desaturase gene in antisense orientation and was included so that silencing could be monitored. Agrobacterium tumefaciens strain LB4404 carrying the helper plasmid pSoup was transformed with constructs PGR106 (empty vector control), PVX. PDS, or PVX. CKP, and used to infect tuberising potato plants in soil by agroinfiltration of leaves.

Plants inoculated with the viral vector showed no viral symptoms, and plants inoculated with PVX. CKP remained indistinguishable from the empty vector control plant as the haulms senesced (a cytokinin-sensitive process) . Tubers were harvested and weighed individually. No difference was detected in the number and fresh mass of tubers per plant. Periderm and storage pith samples were cut from a single tuber for each plant at harvest, snap frozen in liquid N ₂ and stored at 80 ⁰ C; the other tubers were used to assess dormancy. Roughly half the tubers from each plant were given an initial short chill treatment (21d at 4°C) . In our earlier work on ^λ Majestic' we had shown that chill treatment advances the increase in cytokinin levels in tuber buds, contributing to the acceleration of the onset of tuber sprouting. From this we reasoned that silencing StCKPl in tuber tissues might alter the effect of chilling on dormancy, and the chill treatment of ^λ Desiree' tubers was included to test for an effect.

RTP-PCR analysis was carried out on samples from all 11 vector-inoculated plants. RNA was extracted using a Sigma Spectrum Plant Total RNA Kit, and 2μg of RNA was used to produce cDNA, which was then analysed by PCR. Primers within StCKPl were used to amplify a 384 bp fragment of StCKPl. Each reaction included primers for beta-tubulin, which amplified a 609 bp fragment of beta-tubulin as an internal standard. DNA fragments were amplified by 24, 25 and 26 cycles of PCR and separated on a 2% agarose gel. Gel band intensities were quantified with Alphalmager software and results were used from those cycles which showed a doubling of PCR product, as these indicated that accumulation of PCR product was in an exponential phase.

StCKPl was standardised against beta-tubulin for each reaction in exponential phase. This process was repeated for each of three separate cDNA samples produced from extracted RNA.

StCKPl transcripts in storage pith were insufficient for quantitation. Levels in periderm tissue were at least 2OX higher, and here VIGS had been successful. Each plant showed a different degree of silencing, down to one third of control levels. We think this may be the first time that VIGS has been demonstrated quantitatively in potato tuber tissue.

The time elapsed from ^λ lifting' up to 3mm apical sprout length was recorded for each individual tuber, and the mean value for each plant, and for the size classes 0 to 2Og, 21 to 4Og and >40g, determined. The period of dormancy was generally shorter for the smaller tubers, the opposite of previous experience, but the effect was shown by the control plants, therefore if it is real it may be due to the presence of the viral vector.

My original prediction that silencing StCKPl in tuber tissue would extend the duration of dormancy (DD) proved to be wrong; in fact, silencing StCKPl was found to shorten the duration of dormancy. For the plants, tubers given a chill treatment had DD values close to 108d. The mean value for un-chilled tubers of the empty vector control is 14Od, i.e. the chill treatment shortens DD in Desiree by roughly one quarter. For un-chilled tubers from plants in which VIGS was most effective, values of DD were all close to 108d, i.e. the period of dormancy sensitive to chill

treatment has been eliminated. For plants with intermediate levels of silencing, there was an intermediate value for the mean duration of chill-sensitive dormancy

(Fig 12). The quantitative relationship between the abundance of StCKPl transcripts in periderm at the beginning of storage and the extension of endodormancy due to warm storage is striking, particularly in view of the tuber-tuber variance for DD and the fact that the RT-PCR measurements were carried out on tubers not used for DD estimation.

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