Abstract:
The present invention includes modified phytochrome A (PHYA) nucleic acid molecules in which DNA sequences coding for “active site” amino acid residues have been mutated to generate hyperactive phytochromes. In particular; a serine/threonine residue at the hinge between the N- and C-terminal domains as well as at the N-terminal serine/threonine cluster of phytochromes (e.g., serine-598 and serine-7 in oat phytochrome A) for (a) Pr/Pfr-dependent phosphorylation and (b) dephosphorylation by a phytochrome phosphatase (PP2A) was substituted with alanine. (c) In addition, amino acid residues within the phytochrome chromophore pocket are mutated to generate the bathchromic shift of the Pr-absorption band of both wild type and above-mentioned mutant phytochromes. The plants with the bathchromically shifted absorption spectrum are expected to respond to the canopy and shade conditions for growth and greening responses to far-red light with greater efficiency than are the wild type plants with normal absorption band maxima. These mutative modifications confer hyperactivity to the far-red light responsive phytochromes A. Thus, the biological activity of the modified oat PHYA was shown to be hyperactive compared to wild type PHYA, characterized by its ability to reduce internode elongation of adult plants. Overexpression of the phytochrome phosphatase exhibits a suppressed growth with shorter internodes and belated flowering, qualitatively consistent with the phenotype of a ser598ala mutant oat phytochrome. The invention also includes plants having at least one cell expressing the modified PHYA, vectors comprising at least one portion of the modified PHYA nucleic acids, and methods using such vectors for producing plants with reduced stature.

Description:
FIELD OF THE INVENTION  
         [0001]    This invention relates to the concept of functionally hyperactive red/far-red light sensor genes such as a modified phytochrome A (PHYA) nucleic acid molecule of which a serine/threonine amino acid at the hinge region between the amino- and the carboxy-domains and at the N-terminal serine/threonine cluster of phytochrome for Pr/Pfr-dependent phosphorylation/dephosphorylation was substituted with alanine. The phytochrome A functions as the photoreceptor in far-red wavelength light in mediating the suppression of shade avoidance and the greening of leaves. These modified phytochromes lacking critical serine/threonine residues for phosphorylation are hyperactive under far-red light. Another group of the modified phytochromes with bathochromic shift in their Pr-absorption band greatly is to provide an enhanced far-red sensitivity of plants growing under canopy and shade conditions. The present invention also provides the methods and processes for generating transgenic higher plants transformed with the said nucleic acid molecule to engineer internode elongation of economically important crop plants.  
         BACKGROUND OF THE INVENTION  
         [0002]    Phytochrome is a photoreceptor that control diverse aspects of growth and development in higher plants. Upon irradiation, the photoreceptor undergoes reversible interconversion between biologically inactive, red-absorbing phytochrome (Pr) and biologically active, far-red light absorbing phytochrome (Pfr) that enables it act as a molecular light switch. Photoconversion into Pfr form by red light treatment triggers its nuclear translocation from cytosol, initiating signaling that alters gene expression and thereby growth and development of plants. There are two photoisomers, red light (λmax=660 nm) absorbing form (designated to Pr) and far-red light (λmax=730 nm) absorbing form (designated to Pfr). Particularly, the absorption spectra of phytochrome are near the spectrum of canopy (FIG. 1, Neff et al, 2000). This spectral property shows it is directly related to shade avoidance. The initiation of shade avoidance depends on low R (red): FR (far red light) ratio. Low R: FR ratio accelerates not only the shade avoidance reaction that involves hypocotyls elongation, but also early flowering that causes immature fruit developments (Smith &amp; Whitelam, 1997).  
           [0003]    The photoreceptor consists of a 116-127 kDa apoprotein and a covalently attached linear tetrapyrrole chromophore. In plants, the apoproteins are encodes by a small gene family, e.g., five members PHYA-E in Arabidopsis. Molecular genetic analysis revealed that individual members of phytochromes play overlapping but distinct physiological roles. PHYA, a type 1 photo-labile phytochrome, controls very low fluence response and FR-high irradiance response, while type 2 phytochrome, encoded by PHYB-E, abundant in light-grown tissues, regulates low fluence responses (Quail et al., 1995; Neff et al., 2000).  
           [0004]    Previously, oat PhyA was shown to undergo post-translational modification after red-light treatment, including phosphorylation at serine 598 th  residue (Lapko et al., 1999). The Pfr-specific phosphorylation at serine 598 th  residue suggested a regulatory role of this residue on photo-sensory signalling. To test the possibility, in the present invention, we performed site-directed mutagenesis with oat PHYA, substituting serine 598 th  to alanine (designated S598A PHYA in the invention). The biological activity of mutated PHYA was compared with wild type PHYA by overexpression into phyA-null mutant of Arabidopsis. Under FR light condition, both wild type PHYA and S598A PHYA could complement phyA-deficient mutant, showing FR-high irradiance response. However, at adult stage, transgenic Arabidopsis plants overexpressing S598A PHYA exhibited shortened internode in adult plants and shortened petiole, whereas transgenic plants overexpressing wild type PHYA did not show any noticeable defect in adult morphology. Overexpression of PP2A gene resulted in a suppressed internode phenotype similar to that of S598A mutant phytochrome. Thus, we include in the invention the overexpression of PP2A gene as being equivalent to bona fide hyperactive phytochrome by keeping it dephosphorylated in vivo. These results indicate that S598A PHYA is more biologically active than wild type PHYA at least in the regulation of internode elongation.  
           [0005]    Serine-to-alanine substitutions at the N-terminal serine/threonine cluster in phytochromes result in hyperactive phytochromes in Arabidopsis thaliana (Stockhaus et al., 1992). Among the N-terminal serine residues, serine-7 is the only residue in the cluster that is specifically autophosphorylated or phosphorylated by a phytochrome kinase in vivo (Lapko et al., 1997). Thus, S7A mutant phytochrome is a hyperactive phytochrome.  
           [0006]    It has been possible to locate the active site of the autophosphorylating phytochrome A (acting as a “phytochrome kinase”). The PAS-related domain in the C-terminal half of the protein contains active site residues. Mutation or deletion of these residues is expected to result in hyperactivity of phytochrome A in vivo, since such mutants cannot autophosphorylate the protein.  
           [0007]    By using the method of site-directed mutagenesis (Bhoo et al., 1997) and DNA shuffling, we have also generated phytochrome A mutants that absorb far-red shade light more effectively than wilt type. This was achieved by substituting critical amino acid residues (for example, isoleucine-80) within the chromophore binding crevice of phytochrome A. FIG. 1 illustrates how a few nanometer red shift of the Pr-absorption band, so that it can absorb canopy and shade lights several orders of magnitude more effectively in the far-red wavelength than with the overexpression of wild type phytochrome. We propose that the far-red spectral action spectrum for the induction of seed germination (Shinomura et al., 1996) is consistent with the Pr-absorption spectrum of “hot band” or “twisted” chromophore conformation origin, the bathochromic mutant phytochromes are hyperactive in the responses of higher plants to far-red light.  
           [0008]    This invention can be practically applied to control growth and development in general and internode elongation and leaf greening of higher plants in particular (Smith and Whitelam, 1997). The higher plants referred to here are those economically important in agriculture and horticulture. As used herein, the term “economically important higher plants” refers to higher plants that are capable of photosynthesis and widely cultivated for commercial purpose. The term “plant cell” includes any cells derived from a higher plant, including differentiated as well as undifferentiated tissues, such as callus and plant seeds.  
         SUMMARY OF THE INVENTION  
         [0009]    The present invention relates to nucleic acid molecules encoding modified phytochrome A (PHYA) protein of which 598 th  serine amino acid for Pfr-dependent phosphorylation was substituted by alanine. Such nucleic acid molecules preferentially encode a protein with the amino acid sequence as given in SEQ ID NO: 2. The mutant phytochrome A displays hypersensitive biological activity in the response of higher plants to far-red wavelength light.  
           [0010]    The present invention extends to other mutant phytochromes that exhibit similar hyperactivity in the far-red spectral region and under canopy/shade light conditions. Such mutant phytochromes include 7 th  serine-to-alanine mutants, PAS-related domain substitution/deletion mutants, and also the spectral mutants that absorb far-red light effectively.  
           [0011]    Also, provided includes an uninterrupted gene sequence encoding the S598A PHYA, a nucleic acid fragment that can be directly ligated into recombinant DNA constructs, and the S598A PHYA expression vectors that can be readily used to transform cells of higher plants according to the present invention.  
           [0012]    Provided also are transgenic higher plants that are readily accessible to the Agrobacterium-mediated transformation. Overexpression of the S598A PHYA gene results in shortened internodes. These phenotypic traits can be exploited in a way that higher plants of interest harboring the S598A PHYA gene exhibit dwarfism, a very important commercial trait in horticulture and agriculture.  
           [0013]    Therefore, the present invention provides: 1. Nucleic acid molecules encoding a polypeptide of a modified oat phytochrome A (PHYA) of which  598   th  serine amino acid for Pfr-dependent phosphorylation was substituted by alanine, comprising a nucleotide sequence as given in SEQ ID NO: 1. 
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0014]    [0014]FIG. 1 shows the illustration of the bathochromic shift of the Pr-absorption spectrum. Note that the Pr form of phytochrome now strongly absorbs far-red light of wavelength longer than 700 nm.  
         [0015]    [0015]FIG. 2 shows site-directed mutagenesis of oat phytochrome A. The 598 th  serine residue, a target of Pfr-dependent phosphorylation was changed to alanine. After mutagenesis, XbaI digestion was performed to get a correct mutant gene (A). mt1 and mt2 are two different clones after mutagenesis, and wt is oat wild-type phyA gene in the vector. S is Promega 1 kb DNA ladder (G5711). Some DNA sizes of 1 kb DNA ladder are indicated. A XbaI site at 1798 bp was created during the mutagenesis. So, there are 3 fragments in the WT, 2820 bp, 525 bp and 3330 bp, whereas there are 4 fragments in the MT, 1760 bp, 951 bp, 525 bp and 3330 bp. At the bottom of the figure A, 525 bp band was shown. From the results, mt1 clone showed the right restriction pattern and was further confirmation by DNA sequencing. B. DNA sequencing gel showing the changes of bases. WT sequence 5′-AGTT-3′ was changed to 5′GCTC-3′, which changed the Serine at 598 to Alanine.  
         [0016]    [0016]FIG. 3 shows transgene expression of wildtype PHYA and S598A PHYA. A. RT-PCR. The arrow showed the amplified bands of C-terminus DNA fragment of oat phyA (581 bp). B. Western blot analysis. 50 μg of each protein sample was used for this analysis. The arrow showed the protein band of phyA. Lane WT, protein sample from wild-type  Arabidopsis thaliana  (positive control); lane A-, protein sample from wild-type  Arabidopsis thaliana  phyA-201 mutant (negative control); the number represents independent transgenic seed lines of WT and MT; lane S, DNA standard (Gibco 1 kb ladder, 15615-016).  
         [0017]    [0017]FIG. 4 shows FR-high irradiance response of transgenic seedlings. The seedlings were grown on MS media for 4 days in darkness or under FR light. The scale bar indicates 5 mm.  
         [0018]    [0018]FIG. 5 shows adult morphology of transgenic plants. A. The morphology of representative plants grown under longa-day condition for 5 weeks. B. The average heights of plants grown under long day condition for 6 weeks. Each measurement was done with at least 12 plants. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0019]    Phytochromes are the best characterized photoreceptor that regulate diverse aspects of growth and development in higher plants. Upon irradiation, it exhibits interconvertible photo-conversion between biologically inactive Pr (red absorbing phytochrome) form and biologically active Pfr (far-red absorbing phytochrome) form that enables it to act as a molecular light switch (Butler et al., 1959). The activated Pfr triggers downstream signaling that result in diverse photo-responses. Upon Pfr formation after red light absorption, phytochrome undergoes several conformational changes. The Pfr-chromophore is more exposed than the Pr-chromophore (Park et al., 2000). The N-terminal domain is more exposed in the Pr form than in the Pfr form. The hinge region is preferentially exposed in the Pfr form. These conformational changes would trigger downstream signaling events. Phosphorylation is a primary mechanism that transduces signaling in eukaryotes. Phytochrome signaling involves several phosphorylation events. Phytochrome itself exhibited Ser/Thr kinase activity (Yeh and Lagarias, 1997). PKS1, one of the phytochrome interacting factors including PIF3 and NDPK2 have been phosphorylated by phytochrome (Fankhauser, et al., 1999). Interestingly phytochrome is also phosphorylated in a Pfr-dependent manner. The 598 th  Serine residue is preferentially phosphorylated in the Pfr form in vivo (Lapko et al., 1999). In vitro kinase assay showed that the 598 th  serine was shown to be important for the light-regulation of autophosphorylation/phosphotransfer activity of phytochrome. As an effort to characterize the biological role of phosphorylation at 598 th  serine of phytochrome in vivo, we performed site-directed mutagenesis and generated mutant PHYA of which 598 th  serine was substituted by alanine. After generation of transgenic plants that overexpress wildtype PHYA or mutant PHYA using phyA-null Arabidopsis mutant, the phenotypes of transgenic plants were examined.  
         [0020]    Using immunoblot analysis, we identified transgenic lines that overexpress foreign gene, PHYA or mutant PHYA (FIG. 3). Two lines of wildtype PHYA overexpressing lines, designated as WT#4 and WT#6, and several lines of S598A PHYA overexpressing lines were chosen for further analysis.  
         [0021]    To test whether introduced PHYA is biologically functional in Arabidopsis, we grew seedlings under FR light or in the dark. As shown in FIG. 4, Ler wild type showed typical FR-responses, including shortened hypocotyl, expanded cotyledons, while phyA-null mutant exhibited skotomorphogenic development, such as long hypocotyl, closed cotyledons. The WT#4 and WT#6 transgenic seedlings showed typical light-dependent photomorphogenic development. Under the same condition, S598A PHYA transgenic lines complemented phyA null mutant, exhibiting FR-dependent photomorphogenic development. These results indicate that S598A PHYA is functional, complementing phyA-deficiency of phyA-201 mutant in Arabidopsis.  
         [0022]    Previously oat PHYA was shown to be active in several dicot plants (Boylan and Quail, 1989; Boylan and Quail, 1991), mediating FR-HIR. In Arabidopsis, transgenic lines that overexpressing PHYA did not show any effects on adult morphology, while transgenic lines of tobacco and tomato exhibited several agronomic important traits such as dwarfism. When we grew the transgenic Arabidopsis plants that overexpress S598A PHYA, the transgenic lines showed dwarfism, while transgenic lines of PHYA were normal, compared to wild type (FIG. 5). The results suggest that S598A phyA is hyperactive to mediate adult dwarfism in Arabidopsis, compared to wildtype phyA. This trait is a potent agronomical target that can be applied to flowering plants to reduce cell/organ elongation resulting in improved agronomic values  
       EXAMPLES  
       [0023]    Plant Materials and Growth Conditions  
         [0024]    Seeds of pea plant was germinated and grown under sterile condition on the Murashige and Skoog (MS) media. The  Arabidopsis thaliana  ecotype Ler, phyA-201 mutants, and transgenic lines were grown on 0.5×MS medium. All Arabidopsis cultures were maintained in a controlled environment culture room at 26° C., 70% humidity and for the photoperiod of 16 hours. The Arabidopsis transformation was performed according to the simplified floral dip method, a well known technique to the art. For FR-high irradiance response, growth chamber (model E-30LEDI; Percival Scientific, Inc., Boone, Iowa) equipped with FR light-emitting diode was used.  
         [0025]    Enzymatic Treatments of DNA  
         [0026]    DNA manipulations were carried out according to the standard procedures with some modifications whenever required. Restriction enzyme digestions were routinely done in 20 μl reaction volumes with an enzyme of 1-5 units per microgram DNA, and the mixtures were incubated at an appropriate temperature for 1-2 hours. Restriction enzyme digestion buffers used were those supplied by the manufacturer for each particular enzyme, unless specified otherwise. For ligation reactions, DNA fragments, either a digestion mixture or a PCR product, were first separated on 0.8-1.5% agarose gels, depending on the sizes of the DNA fragments of interest, and the desired DNA fragment was purified from the gel piece using either the GENECLEAN II Kit (BIO 101, Vista, USA) or the Gel Extraction Kit (Omega Biotek, Doraville, USA). Ligations were performed usually at the molar ratio of 1:1 to 1:3 in a 10 μl volume using the buffer supplied by the manufacturer, and the mixture was incubated at 13-16° C. for 10 minutes (for sticky-end ligations) or 30 minutes (for blunt-end ligations). T4 DNA ligase and its corresponding ligase buffer (NEB, Beverly, Mass., USA) were routinely used with 5-10 units of ligase in a 10 μl volume reaction. Polymerase chain reaction (PCR) was usually carried out 25 cycles, each with 1 minute denaturation at 94° C., 1 minute annealing at 60° C., and polymerization at 72° C. for 2 minutes per 1000 bases using the Pfu polymerase. For quantitative analysis, PCR was run 15-20 cycles, depending the gene expression levels, using the Taq polymerase (Promega, Madison, Wis.).  
         [0027]    [0027] E. coli  Transformation  
         [0028]    For general cloning purpose,  E. coli  strain XL1-blue was routinely used as host cells for the transformation with plasmid DNAs. The competent  E. coli  cells were prepared in the laboratory and usually had an efficiency of 5×10 −6  to 10 −7  colonies per μg control vector DNA. Three to five microliter of the ligation mixture was usually used to transform 100 μl of the competent  E. coli  cells. After incubation on ice for 20 minutes, the cell-DNA mixture was heat-shocked at 42° C. for 1 minute, and 1 ml of SOC medium was added. The mixture was then gently rotated at 37° C. for 1 hour to render the cells recovered from damage, and 50-300 μl was spread on LB plates containing an appropriate antibiotic. The plates were incubated at 37° C. overnight or until positive colonies were visible.  
         [0029]    Plasmid Isolation and Purification  
         [0030]    Vector DNA was isolated routinely by the alkaline-SDS method from  E. coli  culture. A 1 ml (for high copy number plasmid) or a 10 ml LB-ampicillin culture (for low copy number plasmid) was routinely prepared for the small scale purification of plasmid DNA. For the large scale purification, TB medium (Terrific broth, 47.6 grams of TB mix per liter, Difco, Detroit, USA) which gives higher plasmid DNA yields, instead of LB medium, was used. To prepare plasmid DNA for DNA sequencing and Agrobacterium transformation, those isolated by the alkaline-SDS method was further purified using the Plasmid Miniprep Kit II (Omega Biotek, Seoul, KOREA).  
         [0031]    The Expression of the Genes and Proteins in the Transgenic Plants  
         [0032]    After the screening of the transgenic plants, RT-PCR technique was used to confirm the transcription of the introduced gene. Total RNAs from the transgenic seedlings were prepared by using RNeasy® Plant mini kit (Qiagen, 74903) and followed the standard procedure to generate cDNA by MMRV-reverse transcriptase (Strategene). 5 μg of total RNA was used for the cDNA synthesis. After the synthesis of the cDNA, PCR was performed to confirm the expression of the genes in the transgenic plants. The used primers were 5′-GAATGAAGAACAGATGAAGC-3′ (SEQ ID NO: 3) and 5′-TTGTCCCATTGCTGTTGGAGC-3′ (SEQ ID NO: 4). The products are the C-terminal gene fragments of oat phyA whose size is 581 base pairs. To check the expression of WT and MT proteins and the amounts, the western blot analysis was performed. The preparation of protein samples from the transgenic plants was done as follows: about 4 leaves from each plant were taken off before bolting, put the leaves between the water-soaked Whatman filter papers, and incubated the leaves for at least 12 hours under dark condition. The leave samples were grinded in the microcentrifuge tubes using sea sands and plastic rods. This protein extraction procedure were performed on the ice or in the cold room under the green light condition, and the used buffer for the protein extraction composed of 70 mM Tris (pH 8.3), 35% ethylene glycol, 98 mM (NH 4 ) 2 SO 4 , 7 mM EDTA, 14 mM Sodium metabisulfite, 0.07% polyethyleneimine and 2.8 mM PMSF (all from Sigma except ethylene glycol that is from Fisher). The extracted protein samples were centrifuged at 14,000 rpm and 4° C. for 15 min, and the supernatant were used as protein samples for the western blot analysis. The protein samples were quantified by using Bio-Rad protein assay kit (500-0001), and 50 ug of protein samples were loaded onto the 10% SDS-PAGE gels for the western blot analysis. The protein bands on the SDS-PAGE gel were transferred to PVDF membrane (Hybond-P, Amersham Phamacia Biotech), and the membrane was incubated with oat phyA-specific monoclonal antibody, oat22 and oat25, for 2 hours and developed by using ECL western blotting analysis system purchased from Amersham Phamacia Biotech (RPN 2108). For the detection of Arabidopsis phyA, P25 and mAA7 antibodies were added to the reaction.  
         [0033]    Site-Directed Mutagenesis of S598A Oat PHYA  
         [0034]    The full size of cDNA encoding Avena phytochrome A (phyA) from pFY 122 (Boylan and Quail, 1989) was cloned to pGEM®-11zf(+) (Promega P2411) by digesting with BamHI and EcoRI. After purifying the pGEM®-11zf(+) plasmids containing full-length oat phyA cDNA, the site-directed mutagenesis in order to create Ser598Ala Avena phyA mutant was performed by using GeneEditor™ in vitro site-directed mutagenesis system (Promega Q9280). The oligonucleotide sequence of mutagenic primer for the mutagenesis is phosphorylated-5′-GCGGGAAGCTGCT CTAGA TAACCAGATTGG-3′ (SEQ ID NO: 5). The bold and italic bases are the mutagenized ones from the original sequence 5′-AGTT-3′ (SEQ ID NO: 6) to 5′-GCTC-3′ (SEQ ID NO: 7), and the underlined sequence, 5′-TCTAGA-3′ (SEQ ID NO: 8) is a created XbaI restriction site which is used for the screening of the mutant gene. This new restriction site (XbaI) was introduced by silent mutation near the position to be mutated, allowing rapid and efficient screening for the mutant phyA (Ser598Ala mutant). After the mutagenesis, the mutagenized plasmids were purified and confirmed by XbaI digestion and DNA sequencing. DNA sequencing was done by using Sequenase version 2.0 DNA sequencing kit (Amersham, USB, US70770) with  35 S-ATP.  
         [0035]    DNA Sequencing and Sequence Analysis  
         [0036]    All cDNA and DNA fragments and the junctions of the expression vector constructs were confirmed by direct DNA sequencing on both strands. DNA sequencing was carried out using the ABI PRISM 310 Genetic Analyzer (Perkin Elmer, Foster City, USA) as described in the manufacturer&#39;s manual. For each sequencing run, about 500 ng of plasmid DNA and 2-4 picomoles of 15-17 mer sequencing primer were used. Computer-assisted sequence analysis was performed using the BLAST program (NCBI, USA).  
         [0037]    Gel Electrophoresis of DNA  
         [0038]    Agarose gel electrophoresis of DNA was usually performed using gels with a concentration range of 0.8-1.5%, depending on the size of the DNA fragments to be analyzed, using the TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0). Electrophoresis was performed at a constant voltage rage of 50-200, depending on the amount of DNA loaded onto wells, for a desired time or until DNA fragments were well separated. The gel was stained with 0.5 μg/ml ethidium bromide solution, visualized on an UV transilluminator, and photographed if required.  
         [0039]    Construction of Plant Expression Vectors  
         [0040]    The wild-type (WT) and Ser598Ala mutant (MT) genes were subcloned into the plant transformation vector, pBI121 (Clontech, Cat No. 6018-1: 13 Kb, CaMV 35S promoter etc.). For the subcloning, the vector (pBI121) was digested with BamHI and EcoICRI, and the WT and MT genes in pGEM®-11zf(+) were eluted by sequential enzyme treatment: EcoRI digestion, T4 polymerase treatment for making blunt ended DNA and BamHI digestion. Since the vector and the genes have one blunt end and one cohesive end, they can be ligated and subcloned. After the subcloning and confirmation of the genes in pBI121, the purified plasmids were used for the transformation into phyA deficient  Arabidopsis thaliana . Since the vector has a kanamycin-resistant gene, the seeds having the transformed genes were selected by geminating on the agar plate containing 50 μg/ml kanamycin.  
       REFERENCES  
       [0041]    Bhoo S. H., Hirano T., Jeong H. Y., Lee J. G., Furuya M. &amp; Song P. S. (1997) Phytochrome photochromism probed by site-directed mutations and chromophore esterification. J. Am. Chem. Soc., 119, 11717-11718  
         [0042]    Boylan M. T. and Quail P. H. (1989) Oat phytochrome is biologically active in transgenic tomatoes. Plant Cell, 1, 765-773.  
         [0043]    Boylan, M. T. and Quail, P. H. (1991) Phytochrome A overexpression inhibits hypocotyl elongation in transgenic Arabidopsis. Proc. Natl. Acad. Sci. USA, 88, 10806-10810.  
         [0044]    Fankhauser, C., Yeh, K. C., Lagarias, J. C., Zhang, H., Elich, T. D., and Chory, J. (1999). PKS1, a substrate phosphorylated by phytochrome that modulates light signaling in Arabidopsis. Science 284, 1539-1541.  
         [0045]    Lapko, V. N., Jiang, X. Y., Smith, D. L., and Song, P. S. (1997). Posttranslational modification of oat phytochrome A: Phosphorylation of a specific serine in a multiple serine cluster. Biochemistry, 36, 10595-10599.  
         [0046]    Lapko, V. N., Jiang, X. Y., Smith, D. L., and Song, P. S. (1999). Mass spectrometric characterization of oat phytochrome A: Isoforms and posttranslational modifications. Protein Science, 8, 1032-1044.  
         [0047]    Neff, M. M., Fankhauser, C., and Chory, J. (2000) Light: an indicator of time and place. Genes Dev. 14, 257-271.  
         [0048]    Park, C. M., Bhoo, S.-H. and Song, P.-S. (2000) Inter-domain crosstalk in the phytochrome molecules, Cell Dev. Biol., 11, 449-456.  
         [0049]    Quail, P. H., Boylan, M. T., Parks, B. M., Short, T. W., Xu, Y., and Wagner, D. (1995) Phytochromes: Photosensory perception and signal transduction. Science, 268, 675-680.  
         [0050]    Shinomura, T., Nagatani, A., Hanzawa, H., Kubota, M., Watanabe, M., and Furuya, M. (1996). Action spectra for phytochrome A- and B-specific photoinhibition of seed germination in  Arabidopsis thaliana.  Proc. Natl. Acad. Sci. USA, 93, 8129-8133.  
         [0051]    Smith H. &amp; Whitelam G. C. (1997) The shade avoidance syndrome: multiple responses mediated by multiple phytochromes. Plant, Cell &amp; Environ. 20, 840-844  
         [0052]    Stockhaus, J., Nagatani, A., Halfter, U., Kay, S., Furuya, M., and Chua, N.-H. (1992) Serine-to alanine substitutions at the amino-terminal region of phytochrome A results in an increase in biological activity. Genes Dev. 6, 2364-2372.  
         [0053]    Yeh, K. C., and Lagarias, J. C. (1998). Eukaryotic phytochromes: light-regulated serine/threonine protein kinases with histidine kinase ancestry. Proc. Natl. Acad. Sci. USA 95, 13976-13981.  
     
       
       
         1 
         
           
             8  
           
           
             1  
             3510  
             DNA  
             Avena sp.  
           
            1 

ccggtagcag caggagcgat acacggggta tacgaccgtt gagtggttca attacttgag     60 

gcaggcgatg tcttcctcaa ggcctgcttc cagttcttcc agcaggaacc gccagagctc    120 

ccaggcaagg gtgttagcac agacaaccct tgatgccgag ctcaatgctg aatatgaaga    180 

atctggtgac tcctttgact actccaagct ggttgaagcc cagcgggatg gtccacctgt    240 

gcagcaaggg cggtcggaga aggtcatagc ctacttacag cacattcaga aaggaaagct    300 

aatccaaaca tttggttgcc tgttggccct tgatgagaag agcttcaatg tcattgcgtt    360 

cagcgagaac gcgccagaaa tgcttacaac ggtcagccat gcggtaccca gtgttgatga    420 

tcccccaagg ctggggattg gcaccaatgt acggtctctt ttcagtgacc aaggtgccac    480 

agcactgcac aaggcactag gatttgctga tgtatctttg ctgaatccta tcctagttca    540 

gtgcaagaca tcaggaaagc ctttctatgc cattgttcat cgagcaactg gttgtttggt    600 

ggtagacttt gagcctgtaa agcctacaga atttcctgcc actgctgctg gggctttgca    660 

gtcctacaag cttgctgcca aggcaatatc caagatccag tcattgccag gtggaagcat    720 

ggagatgcta tgcaatactg tggtgaagga agtctttgac cttaccgggt atgatagggt    780 

tatggcttac aagttccatg aagatgacca tggtgaggta ttctccgaaa tcacaaagcc    840 

tggtcttgag ccttatctag gcctgcacta tccagccact gatatccctc aagcagccag    900 

gtttcttttc atgaagaaca aagtacggat gatttgtgat tgccgtgcga gatccataaa    960 

ggtcattgaa gctgaggcac tcccctttga tattagccta tgtggttcag cactcagggc   1020 

accacacagt tgtcaccttc agtatatgga gaacatgaac tcgattgcat cccttgtcat   1080 

ggctgttgtg gttaatgaga atgaagagga tgatgaagct gagtctgaac aaccagcaca   1140 

gcagcagaaa aagaagaaac tatggggcct ccttgtttgc caccatgaga gccctagata   1200 

tgtccctttt ccgctgcgtt atgcttgtga gttcttagca caggtgtttg ctgtccatgt   1260 

caacagggag tttgaattag agaaacagtt gcgtgagaag aacatactga agatgcaaac   1320 

aatgctctct gatatgttgt tccgagaagc ctctcccctg actatcgtat cagggaaccc   1380 

gaatatcatg gacctagtca aatgtgatgg tgctgctctt ctgtatgggg gcaaagtatg   1440 

gcgtctgcgt aatgctccaa cggagtctca gatacatgat atcgccttct ggctatcaga   1500 

tgttcacagg gattccactg gcctgagtac tgacagcctc catgatgctg gctatccagg   1560 

agctgctgct cttggtgata tgatttgtgg aatggcagtg gctaagatca actccaagga   1620 

tattcttttt tggttcaggt cacatacagc tgctgaaatc agatggggag gtgcaaagaa   1680 

tgatccatcg gacatggatg acagcagaag gatgcaccct aggttgtctt tcaaagcttt   1740 

ccttgaagtt gtcaagatga agagcttgcc ttggagtgac tatgaaatgg atgctattca   1800 

ttcattgcaa cttatactgc gagggacact aaatgatgcc agcaagccaa agcgggaagc   1860 

tgctctagat aaccagattg gtgatctaaa acttgatggg cttgctgaac tgcaggccgt   1920 

gaccagtgaa atggttcgtc taatggaaac agcaactgtt ccaatcttgg cagtagatgg   1980 

caatggactg gtcaacgggt ggaatcagaa agcagcggag ttgactgggc taagagttga   2040 

tgatgcaatt ggaaggcaca tacttaccct tgtggaggac tcctctgtac cagttgtcca   2100 

gaggatgcta tatctagctc tgcagggtaa agaagagaag gaagttcgat ttgaggtaaa   2160 

gactcatggc ccgaagaggg atgatggtcc agttatcttg gttgtgaatg cttgtgccag   2220 

tcgggacctt catgatcatg ttgttggagt gtgctttgtt gcccaagata tgactgtcca   2280 

taagttggtg atggacaagt ttactcgggt tgagggtgac tacaaggcga tcattcacaa   2340 

cccgaaccca ctcattcctc ctatatttgg tgctgacgaa tttggatggt gttcggagtg   2400 

gaatgctgca atgaccaagt tgactgggtg gaatagagat gaagtgctcg ataagatgct   2460 

tcttggtgaa gtgtttgaca gtagcaatgc ttcctgccct ttgaagaaca gagatgcatt   2520 

tgtaagtctt tgtgttctta tcaacagtgc attagccggg gaagaaacag aaaaggctcc   2580 

atttggcttc ttcgacagaa gtggaaagta cattgagtgt cttctatcag caaacagaaa   2640 

agaaaatgag ggtggtctca tcactggagt attctgtttt attcatgttg ctagtcatga   2700 

gctgcaacat gcactacagg tgcagcaagc ctcggagcaa acgtcgctaa aaaggctcaa   2760 

ggctttctcc tacatgagac atgcgatcaa caaccctctc tcaggcatgc tctactctag   2820 

aaaagcattg aagaacacag atttgaatga agaacagatg aagcagattc atgttggaga   2880 

taattgtcac caccagataa acaagatact tgcagacttg gatcaagata gcatcaccga   2940 

aaaatctagc tgcttggatt tggagatggc tgaatttctg ttgcaagatg tggtggtggc   3000 

tgctgtaagt caagtactga taacctgcca gggaaaaggg atcagaatct cttgcaacct   3060 

gccagagaga tttatgaagc agtcagtcta tggagatggt gttcgactcc agcagatcct   3120 

ctctgacttc ctgtttattt cagtgaagtt ctctcctgtt ggaggttctg ttgagatttc   3180 

ttccaagctg acaaagaaca gcatcggaga aaaccttcat cttattgacc ttgaacttag   3240 

gatcaagcac caaggattag gagtcccagc agagctcatg gcacaaatgt ttgaggagga   3300 

caacaaggag cagtcagagg agggcttgag cctcctagtt tctagaaacc tgctgaggct   3360 

catgaatggt gatgttcggc atctaaggga agctggtgtg tcaaccttca tcatcaccgc   3420 

tgaacttgct tccgctccaa cagcaatggg acaatgatga agccagtgga agtgtacaac   3480 

ttatggtcat caaatgttct gtttgaattc                                    3510 

 
           
             2  
             1129  
             PRT  
             Avena sp.  
           
            2 

Met Ser Ser Ser Arg Pro Ala Ser Ser Ser Ser Ser Arg Asn Arg Gln 
  1               5                  10                  15 

Ser Ser Gln Ala Arg Val Leu Ala Gln Thr Thr Leu Asp Ala Glu Leu 
             20                  25                  30 

Asn Ala Glu Tyr Glu Glu Ser Gly Asp Ser Phe Asp Tyr Ser Lys Leu 
         35                  40                  45 

Val Glu Ala Gln Arg Asp Gly Pro Pro Val Gln Gln Gly Arg Ser Glu 
     50                  55                  60 

Lys Val Ile Ala Tyr Leu Gln His Ile Gln Lys Gly Lys Leu Ile Gln 
 65                  70                  75                  80 

Thr Phe Gly Cys Leu Leu Ala Leu Asp Glu Lys Ser Phe Asn Val Ile 
                 85                  90                  95 

Ala Phe Ser Glu Asn Ala Pro Glu Met Leu Thr Thr Val Ser His Ala 
            100                 105                 110 

Val Pro Ser Val Asp Asp Pro Pro Arg Leu Gly Ile Gly Thr Asn Val 
        115                 120                 125 

Arg Ser Leu Phe Ser Asp Gln Gly Ala Thr Ala Leu His Lys Ala Leu 
    130                 135                 140 

Gly Phe Ala Asp Val Ser Leu Leu Asn Pro Ile Leu Val Gln Cys Lys 
145                 150                 155                 160 

Thr Ser Gly Lys Pro Phe Tyr Ala Ile Val His Arg Ala Thr Gly Cys 
                165                 170                 175 

Leu Val Val Asp Phe Glu Pro Val Lys Pro Thr Glu Phe Pro Ala Thr 
            180                 185                 190 

Ala Ala Gly Ala Leu Gln Ser Tyr Lys Leu Ala Ala Lys Ala Ile Ser 
        195                 200                 205 

Lys Ile Gln Ser Leu Pro Gly Gly Ser Met Glu Met Leu Cys Asn Thr 
    210                 215                 220 

Val Val Lys Glu Val Phe Asp Leu Thr Gly Tyr Asp Arg Val Met Ala 
225                 230                 235                 240 

Tyr Lys Phe His Glu Asp Asp His Gly Glu Val Phe Ser Glu Ile Thr 
                245                 250                 255 

Lys Pro Gly Leu Glu Pro Tyr Leu Gly Leu His Tyr Pro Ala Thr Asp 
            260                 265                 270 

Ile Pro Gln Ala Ala Arg Phe Leu Phe Met Lys Asn Lys Val Arg Met 
        275                 280                 285 

Ile Cys Asp Cys Arg Ala Arg Ser Ile Lys Val Ile Glu Ala Glu Ala 
    290                 295                 300 

Leu Pro Phe Asp Ile Ser Leu Cys Gly Ser Ala Leu Arg Ala Pro His 
305                 310                 315                 320 

Ser Cys His Leu Gln Tyr Met Glu Asn Met Asn Ser Ile Ala Ser Leu 
                325                 330                 335 

Val Met Ala Val Val Val Asn Glu Asn Glu Glu Asp Asp Glu Ala Glu 
            340                 345                 350 

Ser Glu Gln Pro Ala Gln Gln Gln Lys Lys Lys Lys Leu Trp Gly Leu 
        355                 360                 365 

Leu Val Cys His His Glu Ser Pro Arg Tyr Val Pro Phe Pro Leu Arg 
    370                 375                 380 

Tyr Ala Cys Glu Phe Leu Ala Gln Val Phe Ala Val His Val Asn Arg 
385                 390                 395                 400 

Glu Phe Glu Leu Glu Lys Gln Leu Arg Glu Lys Asn Ile Leu Lys Met 
                405                 410                 415 

Gln Thr Met Leu Ser Asp Met Leu Phe Arg Glu Ala Ser Pro Leu Thr 
            420                 425                 430 

Ile Val Ser Gly Asn Pro Asn Ile Met Asp Leu Val Lys Cys Asp Gly 
        435                 440                 445 

Ala Ala Leu Leu Tyr Gly Gly Lys Val Trp Arg Leu Arg Asn Ala Pro 
    450                 455                 460 

Thr Glu Ser Gln Ile His Asp Ile Ala Phe Trp Leu Ser Asp Val His 
465                 470                 475                 480 

Arg Asp Ser Thr Gly Leu Ser Thr Asp Ser Leu His Asp Ala Gly Tyr 
                485                 490                 495 

Pro Gly Ala Ala Ala Leu Gly Asp Met Ile Cys Gly Met Ala Val Ala 
            500                 505                 510 

Lys Ile Asn Ser Lys Asp Ile Leu Phe Trp Phe Arg Ser His Thr Ala 
        515                 520                 525 

Ala Glu Ile Arg Trp Gly Gly Ala Lys Asn Asp Pro Ser Asp Met Asp 
    530                 535                 540 

Asp Ser Arg Arg Met His Pro Arg Leu Ser Phe Lys Ala Phe Leu Glu 
545                 550                 555                 560 

Val Val Lys Met Lys Ser Leu Pro Trp Ser Asp Tyr Glu Met Asp Ala 
                565                 570                 575 

Ile His Ser Leu Gln Leu Ile Leu Arg Gly Thr Leu Asn Asp Ala Ser 
            580                 585                 590 

Lys Pro Lys Arg Glu Ala Ala Leu Asp Asn Gln Ile Gly Asp Leu Lys 
        595                 600                 605 

Leu Asp Gly Leu Ala Glu Leu Gln Ala Val Thr Ser Glu Met Val Arg 
    610                 615                 620 

Leu Met Glu Thr Ala Thr Val Pro Ile Leu Ala Val Asp Gly Asn Gly 
625                 630                 635                 640 

Leu Val Asn Gly Trp Asn Gln Lys Ala Ala Glu Leu Thr Gly Leu Arg 
                645                 650                 655 

Val Asp Asp Ala Ile Gly Arg His Ile Leu Thr Leu Val Glu Asp Ser 
            660                 665                 670 

Ser Val Pro Val Val Gln Arg Met Leu Tyr Leu Ala Leu Gln Gly Lys 
        675                 680                 685 

Glu Glu Lys Glu Val Arg Phe Glu Val Lys Thr His Gly Pro Lys Arg 
    690                 695                 700 

Asp Asp Gly Pro Val Ile Leu Val Val Asn Ala Cys Ala Ser Arg Asp 
705                 710                 715                 720 

Leu His Asp His Val Val Gly Val Cys Phe Val Ala Gln Asp Met Thr 
                725                 730                 735 

Val His Lys Leu Val Met Asp Lys Phe Thr Arg Val Glu Gly Asp Tyr 
            740                 745                 750 

Lys Ala Ile Ile His Asn Pro Asn Pro Leu Ile Pro Pro Ile Phe Gly 
        755                 760                 765 

Ala Asp Glu Phe Gly Trp Cys Ser Glu Trp Asn Ala Ala Met Thr Lys 
    770                 775                 780 

Leu Thr Gly Trp Asn Arg Asp Glu Val Leu Asp Lys Met Leu Leu Gly 
785                 790                 795                 800 

Glu Val Phe Asp Ser Ser Asn Ala Ser Cys Pro Leu Lys Asn Arg Asp 
                805                 810                 815 

Ala Phe Val Ser Leu Cys Val Leu Ile Asn Ser Ala Leu Ala Gly Glu 
            820                 825                 830 

Glu Thr Glu Lys Ala Pro Phe Gly Phe Phe Asp Arg Ser Gly Lys Tyr 
        835                 840                 845 

Ile Glu Cys Leu Leu Ser Ala Asn Arg Lys Glu Asn Glu Gly Gly Leu 
    850                 855                 860 

Ile Thr Gly Val Phe Cys Phe Ile His Val Ala Ser His Glu Leu Gln 
865                 870                 875                 880 

His Ala Leu Gln Val Gln Gln Ala Ser Glu Gln Thr Ser Leu Lys Arg 
                885                 890                 895 

Leu Lys Ala Phe Ser Tyr Met Arg His Ala Ile Asn Asn Pro Leu Ser 
            900                 905                 910 

Gly Met Leu Tyr Ser Arg Lys Ala Leu Lys Asn Thr Asp Leu Asn Glu 
        915                 920                 925 

Glu Gln Met Lys Gln Ile His Val Gly Asp Asn Cys His His Gln Ile 
    930                 935                 940 

Asn Lys Ile Leu Ala Asp Leu Asp Gln Asp Ser Ile Thr Glu Lys Ser 
945                 950                 955                 960 

Ser Cys Leu Asp Leu Glu Met Ala Glu Phe Leu Leu Gln Asp Val Val 
                965                 970                 975 

Val Ala Ala Val Ser Gln Val Leu Ile Thr Cys Gln Gly Lys Gly Ile 
            980                 985                 990 

Arg Ile Ser Cys Asn Leu Pro Glu Arg Phe Met Lys Gln Ser Val Tyr 
        995                1000                1005 

Gly Asp Gly Val Arg Leu Gln Gln Ile Leu Ser Asp Phe Leu Phe Ile 
   1010                1015                1020 

Ser Val Lys Phe Ser Pro Val Gly Gly Ser Val Glu Ile Ser Ser Lys 
1025               1030                1035                1040 

Leu Thr Lys Asn Ser Ile Gly Glu Asn Leu His Leu Ile Asp Leu Glu 
               1045                1050                1055 

Leu Arg Ile Lys His Gln Gly Leu Gly Val Pro Ala Glu Leu Met Ala 
           1060                1065                1070 

Gln Met Phe Glu Glu Asp Asn Lys Glu Gln Ser Glu Glu Gly Leu Ser 
       1075                1080                1085 

Leu Leu Val Ser Arg Asn Leu Leu Arg Leu Met Asn Gly Asp Val Arg 
   1090                1095                1100 

His Leu Arg Glu Ala Gly Val Ser Thr Phe Ile Ile Thr Ala Glu Leu 
1105               1110                1115                1120 

Ala Ser Ala Pro Thr Ala Met Gly Gln 
               1125 

 
           
             3  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            3 

gaatgaagaa cagatgaagc                                                 20 

 
           
             4  
             21  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            4 

ttgtcccatt gctgttggag c                                               21 

 
           
             5  
             30  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            5 

gcgggaagct gctctagata accagattgg                                      30 

 
           
             6  
             4  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            6 

agtt                                                                   4 

 
           
             7  
             4  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            7 

gctc                                                                   4 

 
           
             8  
             6  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            8 

tctaga                                                                 6