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 
     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 
     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). 
     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). 
     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. 
     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. 
     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. 
     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 wild 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. 
     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 
     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. 
     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. 
     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. 
     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. 
     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 
         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. 
       FIG.  2 A and  FIG. 2B  show 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 (G571 1). Some DNA sizes of 1 kb DNA ladder are indicated. A XbaI site at 1798bp was created during the mutagenesis. So, there are 3 fragments in the WT, 2820bp, 525bp and 3330bp, whereas there are 4 fragments in the MT, 1760bp, 951bp, 525bp and 3330bp. At the bottom of the  FIG. 2A , 525bp band was shown. From the results, m1 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. 
       FIG.  3 A and  FIG. 3B  show transgene expression of wildtype PHYA and S598A PHYA. FIG.  3 A. RT-PCR. The arrow showed the amplified bands of C-terminus DNA fragment of oat phyA (581bp). FIG.  3 B. Western blot analysis. 50 ug 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). 
         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. 
       FIG.  5 A and  FIG. 5B  show adult morphology of transgenic plants. FIG.  5 A. The morphology of representative plants grown under longa-day condition for 5 weeks. FIG.  5 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 
     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. 
     Using immunoblot analysis, we identified transgenic lines that overexpress foreign gene, PHYA or mutant PHYA (FIG.  3 A and FIG.  3 B). 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. 
     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.    
     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 
     Plant Materials and Growth Conditions 
     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-30LED1; Percival Scientific, Inc., Boone, Iowa) equipped with FR light-emitting diode was used. 
     Enzymatic Treatments of DNA 
     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.). 
       E. coli  Transformation 
     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. 
     Plasmid Isolation and Purification 
     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). 
     The Expression of the Genes and Proteins in the Transgenic Plants 
     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. 
     Site-Directed Mutagenesis of S598A Oat PHYA 
     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. 
     DNA Sequencing and Sequence Analysis 
     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). 
     Gel Electrophoresis of DNA 
     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. 
     Construction of Plant Expression Vectors 
     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. 
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