CA2378814A1 - Gene controlling shoot branching in plants - Google Patents

Abstract

The invention relates to a gene that encodes a cytokinin-metabolizing enzyme, designated SPS, that has been isolated from an Arabidopsis mutant, sps. The sps mutant exhibits an abnormal shoot-branching pattern and developmental program. The SPS gene has the nucleotide sequence of SEQ ID NO: 1. Also disclosed are methods for isolating a plant gene that encodes a cytokinin-metabolizing enzyme. Further, the invention includes novel plants transformed with the SPS gene operatively linked to a plant-active promoter as well as methods for altering growth or development of plants containing an SPS gene.

Description

GENE CONTROLLING SHOOT BRANCHING IN PLANTS
Back_c~ound of the Invention Field of the Invention This invention relates to a novel plant gene that encodes a protein which is involved in the control of plant growth and developmental hormones. More particularly the invention relates to a novel gene that encodes an enzyme involved in cytokinin metabolism and to methods for altering plant growth and development.
Background Technology Modulation of plant hormone levels is important for plant growth and development, as well as for environmental adaptation. This regulation can be achieved via the regulation of hormone synthesis and/or metabolism.
Plants exhibit a wide range of aerial architecture. One of the characteristics that determines the overall plant structure is the shoot-branching pattern. Shoot branching is initiated by the formation of lateral meristem in the leaf axils. In some plant species the growth of the lateral meristem is inhibited by the primary floral shoot, a phenomenon generally known as apical dominance. Cytokinin and auxin are classical plant hormones that play a major role in controlling such developmental process (1). (A
bibliography is provided at the end of the written description.) Auxin represses the outgrowth of the lateral bud, whereas cytokinins stimulate growth.
Imbalance in the two hormone levels causes growth defects. Besides the release of lateral buds from apical dominance, cytokinins also play several other important roles in plant growth and development. These include the delay of leaf senescence, inhibition of root growth and, together with auxin, promotion of cell division and shoot formation in tissue culture. This information is mostly derived from indirect evidence of exogenous application of cytokinins and from plants transformed with the bacterial cytokinin biosynthesis gene, IPT, which encodes isopentenyl transferase (2,3).
Thus, knowledge of biosynthesis, metabolism, distribution, perception and signal transduction of cytokinins .in planta is poorly understood. To this end, in the past few years, a number of cytokinin related mutants have been isolated from ~irabidopsis (4-9).
Several selection schemes have been employed to select for different classes of mutants, in an attempt to dissect mechanisms by which cytokinins regulate plant growth and development. Nevertheless, the molecular nature of the corresponding genes is largely unknown and the types of mutants isolated are mainly restricted by the design of the selection schemes.

Summary of the Invention A gene, designated SPS, has been isolated from an Arabidopsis mutant, sps, which exhibits an abnormal shoot-branching pattern and developmental program. The Arabidopsis SPS gene has the nucleotide sequence shown in SEQ ID N0:1.
SPS has been shown to encode a cytochrome P450, which has been assigned as CYP79F1. Phenotypic abnormalities of the sps mutant and analysis of its hormone levels indicate that SPS encodes an enzyme involved in cytokinin metabolism. Accordingly, in one aspect, the present invention involves a method for altering growth or development of a plant containing a SPS gene which comprises modulating expression of the SPS gene.
Tn a further aspect, the invention provides novel plants transformed with the SPS gene operatively linked to a plant-active promoter. Also embodied within the invention are novel plants exhibiting enhanced cytokinin levels, which are due to suppression of the SPS gene. Suppression of SPS is achieved, for example, by transforming a plant with a nucleic acid encoding an SPS antisense or ribozyme operatively linked to a plant-active promoter. The plant-active promoters employed in these embodiments may be a constitutive promoter, an inducible promoter or a tissue specific promoter, as are well known to those skilled in plant molecular biology.
The CYP79 coding sequences are widely conserved among plants of different genera and species. Thus, the Arabidopsis SPS gene and primers and probes having about 8 or more nucleotides derived from its sequence are useful in methods for preparing an isolated SPS
gene of a plant of a different species.
These and other aspects and advantages of the invention in accordance with the scope of the claims will be apparent to those skilled in the art.
Brief Description of the Drawings Figure 1 shows a wild-type and sps mutant of Arabi dopsis thalisana.
Figure 2 shows the location of cytochrome P450 genes, as well as donor T-DNA and Ds insertion sites in sps alleles 1-5.
Detailed Description of the Invention The SPS gene has been discovered through studies of Arabiclopsis transposon insertion mutant designated supershoot ("sps"). The sps mutant was selected from a collection of gene trap Ds insertion lines (10) in Arabidopsis thaliana ecoptype Wassilewskija (Ws).
The phentotypic abnormalities and elevated cytokinin levels in the sps mutant indicate that the mutant is defective in the regulation of cytokinin levels. It was postulated that the Ds element had transposed to an insertion site of a gene involved in cytokinin metabolism, thereby inactivating the gene to cause cytokinin accumulation.
Because the putative gene was tagged with the Ds element, a fragment of genomic DNA flanking the Ds element from a mutant plant was amplified by TAIL-PCR

(17) for detailed analysis. A search of the Arabidopsis thaliana genomic database revealed that the flanking sequences were identical to the genomic sequences from chromosome l, GenBank GI 4887257;

5 GenBank accession number AC 006341. The donor T-DNA
locus used in the transposon-tagging experiment was also found to be within the same clone. A comparative study between the positions of the donor and re-insertion sites indicated that, in this mutant (designated sps-1 allele, because, as described below, additional alleles also have been found), the element transposed 8.5 kb away from the donor site into the ' coding region of a putative cytochrome P450, assigned previously as CYP79F1 (AC006341, 63874..66127). The PCR
fragment was then used as a probe to screen Arabidopsis thal.iana leaves and a stem cDNA library (18). The longest SPS cDNA clone isolated from the screen was 1.38 kb long. This cDNA was determined to be a partial clone, truncated at the 5' end. Available genomic and EST sequences corresponding to the SPS gene (19) predicted that the start codon of the open reading frame was located 442 by before the beginning of the isolated cDNA. This prediction was supported by the information derived from the SPS closely related gene, which exhibits 89% sequence identity to the SPS gene (see below). The sequence of the full length SPS cDNA
was reconstituted. It is 1.8 kb long and has the sequence shown in SEQ ID N0:1. The SPS gene is predicted to encode 537 amino acids, with a calculated molecular mass of 61.5 kD.
The number of closely related sequences within the CYP79F subfamily was estimated by genomic southern blot analysis (20). In the Arabidopsis genome, only two fragments hybridized to the SPS probe under low stringency, suggesting that there are only two related members in this sub-family. A search of the Arabidopsis database revealed that the closely related sequence locates just next to the SPS gene and has been assigned as CYP79F2 (AC006341, 60246..62290). Locations of the two Cytochrome P450s as well as the donor T-DNA are shown in Fig. 2. To date, 13 members in the CYP79 family have been identified in plant (seven members from Arabidopsis~ one from Sorghum bicolor; one from Sinapis alba; two from Manihot esculenta; two from Triglochin maritima). Among these 13 members, 6 members have been studied in more detail. CYP79A1 from Sorghum bicolor (21) , CYP79B1 from Sinapsis alba (22) , CYP79E1 and CYP79E2 (43) from Triglochin maritima are shown to catalyze the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of plant secondary metabolites, the cyanogenic glucosides.
CYP79B2 and CYP79B3 from Arbidopsis are shown to catalyze the conversion of tryptophan to indole-3-acetaldoxime, a precursor to IAA and indole glucosinolate (44).
To confirm whether CYP79F1 corresponds to the sps mutation, several additional sps alleles caused by independent Ds insertion were isolated. These mutant alleles were readily obtained due to the proximity of the donor T-DNA and the SPS gene. Four more sps alleles, designated sps-2 to sps-5, were isolated from independent insertion lines. Each allele exhibited overall phenotype similar to the sps-1. Genomic Southern blot analysis confirmed that each line contained only a single Ds element in the genome. The Ds-flanking DNA fragment from each allele was isolated and analyzed. All of these alleles contained Ds insertions within the coding region of the CYP79F1 gene. The positions of the Ds elements that disrupt the gene function in the 5 mutant alleles are shown in Fig 2. Taken together, these results indicate that SPS
gene is tagged with Ds element.
There are several sequence motifs conserved among members of the cytochrome P450 and are reported to be important for the protein function. These include the heme-binding domain essential for catalysis, N-terminus hydrophobic region essential for membrane association and a proline/glycine-rich region for proper protein assembly (23, 24). The heme-binding domain of the CYP79 family diverges from other P450s but is conserved among members in the same family. A CYP79 family specific heme binding consensus sequence is defined as SFSTG(K/R)RGC(A/I)A (22). This unique sequence was also conserved in the SPS gene. The N-terminal hydrophobic region and the proline/glycine-rich region are also present.
Control or modification of plant growth and development can be achieved by modulating expression of the SPS gene. Cytokinin levels can be increased by suppressing expression of the gene. Such increased levels of cytokinins enhance lateral shoot formation (27, 28). In contrast, over-expression of the SPS gene using sense constructs under the control of strong promoters suppress lateral shoot formation.
Furthermore, the aerial architecture of a plant can be altered within a defined region by controlling the location and/or timing of expression of the SPS
gene. For example, the modification that leads to only an increase of lateral branches may be achieved by reducing the SPS expression level at the site of bud initiation, with the use of specifically expressed or tissue-specific promoters. Such promoters are known and can be readily isolated by various technologies such as gene trapping (25).
Suppression of SPS expression can be accomplished in a variety of ways known in the art. Antisense technology can be conveniently used. To accomplish antisense expression, a nucleic acid segment from the SPS gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed. The expression cassette is then transformed into plants and the antisense strand of RNA
is produced. In plant cells, it has been suggested that antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the gene of interest (29, 30).
The nucleic acid segment to be introduced generally will be substantially identical to at least a portion of the SPS gene. The sequence, however, need not be perfectly identical to inhibit expression. For antisense suppression, the introduced sequence need not be full length relative to either the primary transcription product or fully processed mRNA.
Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments may be equally effective. Normally, a sequence of between about 30 or 40 nucleotides and about full length may be used, though a sequence of at least about 100 nucleotides is preferred, a sequence of at least about 200 nucleotides is more preferred, and a sequence of at least about 400 nucleotides is especially preferred.
Catalytic RNA molecules or ribozymes can also be used to inhibit expression of the SPS gene. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of 15~ ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.
A number of classes of ribozymes have been described (31). One class of ribozymes is derived from a number of small circular RNAs which are capable of self-cleavage and replication in plants. The RNAs replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples include RNAs from avocado sunblotch viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, Salanum nodiflorum mottle virus and subterranean clover mottle virus.
Enhancement of SPS expression may be accomplished by transforming a plant with. an SPS gene operatively linked to a plant-active promoter. Techniques for incorporating DNA into a plant cell so as to achieve expression in the cell are well known. These techniques are applicable to incorporation of the SPS
gene and to incorporation of antisense or ribozyme constructs.
Generally, such techniques involve inserting the 5 nucleic acid into a DNA expression vector. Such vector advantageously contains the necessary elements for the transcription and translation of the inserted protein coding sequences and one or more marker sequences to facilitate selection of transformed cells or plants.

10 A number of plant-active promoters are known in the art and may be used to effect expression of the desired nucleic acid sequences. Suitable promoters include, for example, the nos promotor, the small subunit chlorophyll A/B binding polypeptide, the 35S
promotor of cauliflower mosaic virus, as well as promoters isolated from the SPS gene. The promoter rnay be isolated from the type of plant to be transformed.
The 35S or actin promoters may also be used for isolated cDNA clones. These are also useful to test overexpression of the gene. Alteration of the SPS gene expression in defined regions of the plant could be achieved by using specifically expressed promoters.
Such promoters can be readily isolated by various technologies such as gene trapping (25).
Alternatively, several inducible promoters, such as GVG, GVGEc, ER-C1 systems, have been described (45).
These inducible promoters can be used to turn on or turn off expression of a transgene.
Once the nucleic acid has been cloned into an expression vector, it is ready to be transformed into a plant cell. The term plant cell includes any cell derived from a plant; this includes undifferentiated tissues such as callus and suspension cultures, as well as plant seeds, pollen or plant embryos. Plant tissues suitable for transformation include leaf tissues, root tissues, meristems, protoplasts, hypocotyls cotyledons, scutellum, shoot apex, root, immature embryo, pollen, and anther.
One technique of transforming plants with the SPS
gene in accordance with the present invention is by contacting tissue of such plants with an inoculum of bacteria transformed with a vector comprising a nucleic acid in accordance with the present invention.
Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28°C.
Bacteria from the genus Agrobacterium can be' utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogens. Agrobacterium tumefaciens (e. a., strains LBA4404 or EHA105) is particularly useful due to its well-known ability to transform plants.
Another approach to transforming plant cells with the nucleic acid of this invention involves propelling inert or biologically active particles at plant cells.
This technique is disclosed in U.S. Pat. Nos.
4, 945, 050, 5, 036, 006 and 5, 100, 792 all to Sanford et.
al., which are hereby incorporated by reference.
Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the nucleic acid of interest. Biologically active particles (e. a., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced) can also be propelled into a plant cell tissue.
Another method of transforming plant cells is the electroporation method. This method involves mixing the protoplasts and the desired nucleic acid and forming holes in the cell membranes by electric pulse so as to introduce the DNA in the cells, thereby transforming the cells. This method currently has high reproducibility and various genes have been introduced into monocotyledons, especially rice plants by this method (32-34).
Similar to the electroporation method is a method in which the desired gene and protoplasts are mixed and the mixture is treated with PEG, thereby introducing the gene into the protoplasts. This method is different from the electroporation method in that polyethylene glycol ("PEG") is used instead of the electric pulse (35-37).
Other methods include 1)culturing seeds or embryos with nucleic acids (38); 2) treatment of pollen tube (39); 3) liposome method (40, 41); and 4) the microinjection method (42).
Known methods for regenerating plants from transformed plant cells may be used in preparing transgenic plants of the present invention. Generally, explants, callus tissues or suspension cultures can be exposed to the appropriate chemical environment (e~a., cytokinin and auxin) so the newly grown cells can differentiate and give rise to embryos which then regenerate into roots and shoots.
Sequences of cytochrome P450 family of proteins, including CYP79F1, are conserved among a wide variety of plant genera and species (21, 22, 43, 44).
Accordingly, the Arabidopsis SPS gene described herein may conveniently be used directly for affecting growth and development of plants, or may be used to prepare SPS genes and gene constructs (including antisense constructs) from other species. Primers and probes useful for identifying such SPS genes, optionally labeled with one or more detectable nucleotides, consist of a nucleic acid having a sequence that is the same as or is complementary to a sequence of any 8 or more, preferably 13 or more, contiguous nucleotides of SEQ ID NO:1. As used herein, "same as" refers to the sequence, recognizing that RNA sequences contain ribonucleotides that correspond to deoxyribonucleotides of DNA. Such primers and probes can be used in a method for isolating an SPS gene by, for example, amplification and/or probing, as is well known in the art.
The invention is further illustrated by the following examples, which are not intended to be limiting.
Example 1 Characterization of SPS Mutant Morpholoav The phenotype of sps mutants is not detectable in young seedlings, but pleiotropic developmental defects can be noticed after plants produce adult leaves. See Figure 1. In sps mutant plants, leaf shape is more serrate and leaf vasculature is less well developed relative to wild type plants. In Arabidopsis, the pattern of vasculature in the cotyledon is simple with only a central vein and two or three interconnecting side branches. The pattern becomes more complex in rosette leaves, with the greatest complexity seen in the late adult leaves. In sps mutants, the pattern of the vasculature in the rosette leaves is much less complex than that in wild type leaves. In addition, the veins of the adult leaves become strikingly prominent, appearing dark green, compared to wild type. When chlorophyll contents were determined in mature leaves, the mutant plants also contain higher level of chlorophyll (11). The severity of these vegetative phenotypes varies depending on growth conditions. The phenotypes can be significantly delayed by growing plants on the synthetic medium.
The phenotypic defects in the sps mutants become more pronounced after the plants start to flower. The sps mutant has the same rate of leaf initiation during vegetative stage and the timing of the transition from vegetative to reproductive phase does not differ from wild type. The sps floral shoot has reduced internode elongation and displays a loss of apical dominance (Fig 1A). In wild type Arabidopsis plants, the number of lateral floral shoots developing from rosette leaves varies, commonly less than 5, depending on the ecotype.
However, mutant plants, which also display a delay of senescence, continue to produce lateral floral shoots from both the axials of rosette and cauline leaves resulting in several hundred shoots per plant after 4-5 months (Fig 1B). Sps mutants display a reduced number 5 of floral organs and are defective in floral development. Petals are usually reduced or missing.
The number of sepals and stamens are also reduced in some flowers. Anthers do not usually release pollen grains and the stigmas remain underdeveloped.
10 Consequently, the mutant has a very low seed production.
C_ytokinin Levels The physiological changes in the aerial part of the sps mutant plants are strikingly similar to the 15 effects of cytokinin caused by exogenous application of hormone and is characteristic of transgenic plants exhibiting cytokinin overproduction (3). These physiological changes include the release of lateral buds from apical dominance, the increase in bud initiation, the delay of leaf senescence as well as the increase in chlorophyll content. Based on these observations, levels of free zeatin, which is the major active cytokinin in Arabidopsis (5,6) were determined.
The sps mutant has an average of three times more active zeatin than wild type (12). The phenotype of sps suggests that increased cytokinin level is likely to be the primary defect responsible for these changes.
Transgenic plants expressing the bacterial IPT gene, which leads to the morphological changes, were mostly reported to exhibit very high cytokinin levels, 50 folds or more (13-15). This dramatic increase in cytokinin is now considered to be non-physiological.

It has been shown that relatively minor changes in cytokinin level, two to sevenfold, are sufficient to cause the same developmental alterations (15,16).
Results from sps mutant studies indicated that, indeed, small fluctuations of cytokinin levels can induce developmental changes in planta. The Arabidopsis mutant altered meristem program (amply has also been shown to have elevated level of endogenous cytokinin (5,6). Even though sps and ampl have loss of apical dominance, reduced fertility and delay senescence, they differ significantly with respect to other developmental programs. Other ampl phenotypes, such as polycotyiy, faster rate of leaf initiation and abnormal phyllotaxy, have not been observed in sps, or in transgenic plants conferring cytokinin overproduction.
On the other hand, the reduced number of floral organs and the characteristic of the leaf veins, appearing dark green, found in sps mutants are not observed in the ampl mutant. To date, there is no molecular information on the structure and function of the AMP1 gene.
SPS Expression Pattern Monitored b« Gene Trap Ds Element Information about the gene action was derived from the study of temporal and spatial expression of the gene in the sps-2 allele. In this allele, a GUS
reporter gene was incorporated between the ends of the Ds element. Expression of the GUS reporter was controlled by the natural SPS promoter in these constructs (25). The expression pattern of SPS as exhibited by the GUS reporter correlates well with the morphological abnormalities observed in the aerial parts. Expression is first detected in the seedling 4-5 days after germination at the branching area of the vascular tissue that lies at the base of the shoot apical meristem. A few days later, the expression level is more intense and found in the vascular tissue in both the hypocotyl and the cotyledons. Expression pattern in the leaf vasculature appears to be developmentaly regulated. It is first observed only in the mid vein in the young leaves and then, when the leaves are more mature, in the finer veins. Expression of SPS gene is restricted solely to the aerial part of the plant which continues from the vegetative phase to reproductive phase. In the floral shoots, it expresses in the vascular tissue of the stem and cauline leaves, with the strongest intensity at the base of the cauline leaves, and at the receptacle of the flower and silique. No SPS expression is observed in the root system either in the seedling stage or in the adult plant. These results suggest that SPS localizes the modulation of cytokinin levels, which in turn control development in defined regions of the plant. SPS
expression levels observed in mutant plants are much stronger than that observed in heterozygous plants exhibiting wild type phenotype. These data imply that expression of SPS is controlled by a feedback regulation mechanism that can enhance expression level upon disruption of the gene. Such a regulation may reflect the ability of the plants to fine tune the level of hormone in response to the fluctuations of internal or external stimuli.
The question of how plants modulate cytokinin spatially and temporally can be deciphered from the analysis of SPS gene expression pattern. This question was difficult to address in the past, due to the fact that plant hormones are present in minuscule amounts in most plant tissues. It has been shown unambiguously that cytokinin is a key factor in promoting bud growth, whereas auxin has an inhibition effect. Therefore the outcome appears to depend on the ratio of the two hormones. Despite this well-established concept of hormone interaction, the mechanism of apical dominance remains unclear. The results obtained from sps strongly suggest that the plant maintains apical dominance in part by attenuating active cytokinin in the meristematic region to the level that it can inhibit the release of bud growth, as well as de novo bud formation.
Example 2 Experimental Description of SPS Cloning To isolate DNA immediately flanking the Ds, about 10 ng genomic DNA from leaf tissue of the mutant plants was used for amplification by TAIL-PCR as previously described (17). The amplified fragments carrying the flanking genomic sequences were isolated by gel electrophoresis and sequenced using standard protocols (20). The PCR amplified fragments were then labeled with 32P-dCTP and used as a probe to screen a cDNA
library made from Arabidopsis leaves and stems (Clontech) as described (20). Phages that hybridized to the PCR fragment were purified and characterized (20 and instructions from manufacturer Clontech). The inserts were then subcloned into. the BZUESCRIPT plasmid (Stratagene) for detailed analysis in accordance with manufacturer's instructions. Plasmids carrying the inserts were sequenced using an automated sequencer (Perkin Elmer AMI 377) and the full length sequence was assembled using the DNA STAR program.

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19. ATTS5112 Gif-SeedA Arabidopsis thaliana cDNA clone YAY778 5', mRNA sequence. Accession F14190.

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SEQUENCE LISTING
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<213> Arabidopsis thaliana <220>
<221> CDS
<222> {1)..(1611) <400> 1 atg agc ttt acc aca tca tta cca tac cct ttt cac atc cta cta gtc 48 Met Ser Phe Thr Thr Ser Leu Pro Tyr Pro Phe His Ile Leu Leu Val ttt atc ctc tcc atg gca. tca. atc act cta ctg ggt cga ata ctc tca. 96 Phe Ile Leu Ser Met Ala Ser Ile Thr Leu Leu GIy Arg IIe Leu Ser agg ccc acc aaa acc aaa gac cga tct tgc cag ctt cct cct ggc cca 144 Arg Pro Thr Lys Thr Lys Asp Arg Ser Cys GIn Leu Pro Pro GIy Pro cca gga tgg ccc atc ctc ggc aat cta ccc gaa cta ttc atg act cgt I92 Pro Gly Trp Pro Ile Leu Gly Asn Leu Pro Glu Leu Phe Met Thr Arg cct agg tcc aaa tat ttc cgc ctt gcc atg aaa gag cta aaa aca gat 240 Pro Arg Ser Lys Tyr Phe Arg Leu Ala Met Lys Glu Leu Lys Thr Asp SUBSTITUTE SHEET (RULE 26) ata gca tgt ttc aac ttt gcc ggc atc cgt gcc atc acc ata aac tcc 288 Ile Ala Cys Phe Asn Phe Ala Gly Ile Arg Ala Ile Thr Ile Asn Ser 8S 90 . 9S
gac gag agc get aga gaa gcg ttt aga gag cga gac gca: gat ttg gca 336 Asp Glu Ser Ala Arg Glu Ala Phe Arg Glu Arg Asp Ala Asp Leu Ala gac cgg cct caa. ctt ttc atc atg gag aca atc gga gac aat tac aaa 384 Asp Arg Pro Gln Leu Phe Ile Met Glu Thr Ile Gly Asp Asn Tyr Lys tca atg gga att tca ccg tac ggt gaa caa ttc atg aag atg aaa aga 432 Ser Met Gly Ile Ser Pro Tyr Gly Glu Gln Phe Met Lys Met Lys Arg gtg atc aca acg gaa att atg tcc gtt aag acg ttg aaa atg ttg gag 480 Val Ite Thr Thr Glu Ile Met Ser Val Lys Thr Leu Lys Met Leu Glu get gca aga acc atc gaa gcg gat aat ctc ata get tac gtt cac tcc S28 Ala Ala Arg Thr Ile Glu Ala Asp Asn Leu IIe Ala Tyr VaI I-Ts Ser atg tat caa. cgg tcc gag acg gtc gat gtt aga gag ctc tcg agg gtt S76 Met Tyr Gln Arg Ser GIu Thr Val Asp Val Arg Glu Leu Ser Arg Val tat ggt tac gca gtg acc atg cga atg ttg ttt gga agg aga cat gtt 624 Tyr Gly Tyr Ala Val Thr Met Arg Met Leu Phe Gly Arg Arg His Val acg aaa gaa aac gtg ttt tct gat gat gga aga cta gga aac gcc gaa 672 Thr Lys Glu Asn Val Phe Ser Asp Asp Gly Arg Leu Gly Asn Ala Glu aaa cat cat ctt gag gtg att ttc aac act ctt aac tgt tta ccg agt 720 Lys His His Leu Glu Val Ile Phe Asn Thr Leu Asn Cys Leu Pro Ser ttt agt cca gcg gat tac gtg gaa cga tgg ttg aga ggt tgg aat gtt 768 Phe Ser Pro AIa Asp Tyr VaI GIu Arg Trp Leu Arg GIy Trp Asn VaI

gat-ggt caa gag aag agg gtg aca gag aac tgt aac att gtt cgt agt 816 SUBSTITUTE SHEET (RULE 26) Asp Gly Gln Glu Lys Arg Val Thr Glu Asn Cys Asn Ile Val Ai-g Ser tac aac aat ccc ata atc gac gag agg gtc cag ttg tgg agg gaa gaa 864 Tyr Asn Asn Pro Ile Ile Asp Glu Arg Val Gln Leu Trp Arg Glu Glu ggt ggt aag get get gtt gaa gat tgg ctt gat acg ttc att acc cta 912 GIy GIy Lys Ala Ala Val Glu Asp Trp Leu Asp Thr Phe Ile Thr Leu aaa gat caa aac gga aag tac ttg gtc aca. cca, gac gaa atc aaa get 960 Lys Asp ~Gln Asn Gly Lys Tyr Leu Val Thr Pro Asp Glu Ile Lys Ala caa tgc gta gaa ttt tgt ata gca gcg att gat aat ccg gca aat aac 1008 GIn Cys Val Glu Phe Cys Ile Ala AIa Ile Asp Asn Pro Ala Asn Asn atg gag tgg aca ctt ggg gaa atg tta aag aac ccg gag att ctt aga 1056 Met GIu Trp Thr Leu GIy Glu Met Leu Lys Asn Pro Glu Ile Leu Arg aaa get ctg aag gag ttg gat gaa gta gtt gga aga gac agg ctt gtg 1104 Lys Ala Leu Lys Glu Leu Asp Glu Val Val Gly Arg Asp Arg Leu Val caa. gaa tca gac ata cca aat cta aac tac tta aaa get tgt tgt aga 1152 Gln Glu Ser Asp Ile Pro Asn Leu Asn Tyr Leu Lys Ala Cys Cys Arg gaa aca ttc aga att cac cca agt get cat tat gtc cct tcc cat ctt 1200 Glu Thr Phe Arg Ile Isis Pro Ser Ala His Tyr Val Pro Ser His Leu gcg cgt caa gat acc acc ctt ggg ggt tat ttc att ccc aaa ggt agc 1248 Ala Arg Gln Asp Thr Thr Leu Gly Gly Tyr Phe Ile Pro Lys Gly Ser cac att cat gta tgc cgc cct gga cta ggt cgt aac cct aaa ata tgg 1296 His Ile His Yal Cys Arg Pro Gly Leu Gly Arg Asn Pro Lys Ile Trp aaa gat cca. ttg gta tac aaa ccg gag cgt cac ctc caa gga gac gga 1344 Lys Asp Pro Leu VaI Tyr Lys Pro GIu Arg His Leu GIn GIy Asp Gly SUBSTITUTE SHEET (RULE 26) atc aca aaa gag gtt act ctg gtg gaa aca gag atg cgt ttt gtc tcg 1392 Ile Thr Lys Glu Val Thr Leu Val Glu Thr Glu Met Arg Phe Val Ser ttt agc acc ggt cga cgt ggc tgc atc ggt gtt aaa gtc ggg acg atc 1440 Phe Ser Thr Gly Arg Arg Gly Cys Ile Gly Val Lys Val Gly Thr Ile atg atg gtt atg ttg ttg get agg ttt ctt caa ggg ttt aac tgg aaa 1488 Met Met Val Met Leu Leu Ala Arg Phe Leu GIn GIy Phe Asn Trp Lys ctc cat ca.a gat ttt gga ccg tta agc ctc gag gaa gat gat gca tca. 153 6 Leu Ids Gln Asp Phe Gly Pro Leu Ser Leu Glu Glu Asp Asp Ala Ser ttg ctt atg get aaa cct ctt cac ttg tcc gtt gag cca cgc ttg gca 1584 Leu Leu Met Ala Lys Pro Leu His Leu Ser Val Glu Pro Arg Leu Ala cca aac ctt tat cca aag ttc cgt cct taa 1614 Pro Asn Leu Tyr Pro Lys Phe Arg Pro <2I0> 2 <211> 537 <212> PRT
<213> Arabidopsis thaliana <400> 2 Met Ser Phe Thr Thr Ser Leu Pro Tyr Pro Phe His Ile Leu Leu Val Phe Ile Leu Ser Met Ala Ser Ile Thr Leu Leu Gly Arg Ile Leu Ser Arg Pro Thr Lys Thr Lys Asp Arg Ser Cys Gln Leu Pro Pro GIy Pro Pro Gly Trp Pro Ile Leu Gly Asn Leu Pro Glu Leu Phe Met Thr Arg SUBSTITUTE SHEET (RULE 26) WO 01/90388 ~ PCT/SG00/00075 Pro Arg Ser Lys Tyr Phe Arg Leu Ala Met Lys Glu Leu Lys Thr Asp Ile Ala Cys Phe Asn Phe Ala Gly Ile Arg Ala Ile Thr Ile Asn Ser Asp Glu Ser Ala Arg Glu Ala Phe Arg Glu Arg Asp Ala Asp Leu Ala Asp Arg Pro Gln Leu Phe Ile Met Glu Thr Ile Gly Asp Asn Tyr Lys Ser Met Gly Ile Ser Pro Tyr Gly Glu Gln Phe Met Lys Met Lys Arg Val Ile Thr Thr Glu Ile Met Ser Val Lys Thr Leu Lys Met Leu GIu Ala Ala Arg Thr Ile Glu Ala Asp Asn Leu Ile Ala Tyr Val Ids Ser Met Tyr Gln Arg Ser Glu Thr Vai Asp Val Arg Glu Leu Ser Arg Val Tyr Gly Tyr Ala Val Thr Met Arg Met Leu Phe Gly Arg Arg His Val Thr Lys Glu Asn Val Phe Ser Asp Asp GIy Arg Leu Gly Asn AIa GIu Lys His His Leu Glu Val Ile Phe Asn Thr Leu Asn Cys Leu Pro Ser Phe Ser Pro Ala Asp Tyr Val Glu Arg Trp Leu Arg Gly Trp Asn Val Asp Gly Gln Glu Lys Arg Val Thr Glu Asn Cys Asn Ile Val Arg Ser Tyr Asn Asn Pro Ile Ile Asp Glu Arg Val Gln Leu Trp Arg Glu Glu Gly Gly Lys Ala Ala Val Glu Asp Trp Leu Asp Thr Phe Ile Thr Leu SUBSTITUTE SHEET (RULE 26) Lys Asp Gln Asn Gly Lys Tyr Leu Val Thr Pro Asp Glu Ile Lys Ala Gln Cys Val Glu Phe Cys IIe Ala Ala IIe Asp Asn Pro Ala Asn Asn Met Glu Trp Thr Leu Gly Glu Met Leu Lys Asn Pro Glu Ile Leu Arg Lys Ala Leu Lys Glu Leu Asp Glu Val Val Gly Arg Asp Arg Leu Val Gln GIu Ser Asp Ile Pro Asn Leu Asn Tyr Leu Lys Ala Cys Cys Arg Glu Thr Phe Arg Ile His Pro Ser Ala His Tyr Val Pro Ser His Leu Ala Arg Gln Asp Thr Thr Leu Gly Gly Tyr Phe Ile Pro Lys Gly Ser 405 410 ' 415 Ids lle I~'ts Val Cys Arg Pro Gly Leu Gly Arg Asn Pro Lys Ile Trp Lys Asp Pro Leu Val Tyr Lys Pro Glu Arg Ids Leu Gln Gly Asp Gly IIe Thr Lys Glu Val Thr Leu Val GIu Thr Glu Met Arg Phe Val Ser Phe Ser Thr Gly Arg Arg Gly Cys Ile GIy Val Lys Val Gly Thr IIe Met Met Val Met Leu Leu AIa Arg Phe Leu Gln Gly Phe Asn Trp Lys Leu Isis GIn Asp Phe Gly Pro Leu Ser Leu Glu Glu Asp Asp Ala Ser Leu Leu Met Ala Lys Pro Leu His Leu Ser Val Glu Pro Arg Leu Ala Pro Asn Leu Tyr Pro Lys Phe Arg Pro SUBSTITUTE SHEET (RULE 26)

Claims (10)

1. An isolated DNA having the nucleotide sequence of SEQ ID NO:1.

2. A nucleic acid molecule having a sequence that is the same as or is complementary to a sequence of any 8 or more contiguous nucleotides of the DNA of SEQ ID
NO:1.

3. A nucleic acid molecule of claim 2, wherein the sequence is the same as or complementary to a sequence of any 13 or more contiguous nucleotides of the DNA or SEQ ID NO:1.

4. A plant cell transformed with the DNA of claim 1, 2 or 3 under control of a operative plant-active promoter.

5. A method for altering growth or development of a plant containing an SPS gene, which comprises modulating expression of the SPS gene.

6. The method of claim 5, in which SPS expression is reduced by transforming the plant with a nucleic acid operatively linked to a plant-active promoter wherein the nucleic acid encodes an antisense RNA or a ribozyme which inhibits expression of the SPS gene.

7. The method of claim 5, in which SPS expression is increased by transforming the plant with an SPS gene operatively linked to a plant-active promoter.

8. A method for isolating a plant gene that encodes a cytokinin-metabolizing enzyme, which comprises identifying in the genome of said plant a coding sequence, having at least about 80% homology to SEQ ID NO:1; and preparing a nucleic acid having said coding sequence.

9. The method of claim 8, wherein the coding sequence has at least about 90% homology to SEQ ID
NO:1.

10. A method for isolating a plant gene that encodes a cytokinin-metabolizing enzyme, which comprises amplifying a target sequence in the genome of said plant using as primers two different nucleic acid molecules as defined in claim 3; identifying the gene having said coding sequence and preparing a nucleic acid having the coding sequence of said gene.