Abstract:
The present invention includes modified phytochrome A (PHYA) nucleic acid molecules in which Pr absorption spectra have been shifted to longer wavelength (i.e. bathochromism or red-shift). The plants with the bathochromic phytochromes are expected to respond to canopy and shade conditions for growth and development with greater efficiency than the plants with wild-type phytochrome (i.e. suppression of shade avoidance reactions in plants). Since the shade avoidance reactions in plants induce a rapid and dramatic increase in the extension growth of stems and petioles at the expense of leaf growth, storage organ production, and reproductive development, it causes significant losses of crop yields. Thus, the bathochromic phytochromes that utilize the shade light efficiently would suppress the shade avoidance reactions in plants, giving plants the tolerance to shade. In this invention, several bathochromic phytochromes were generated by site-directed mutagenesis in the region of bilin lyase domain in plant PHYA, and their ability to suppress the shade avoidance reactions were examined by transforming the bathochromic phytochromes into PHYA deficient  Arabidopsis thaliana  (ecotype col-0). The transgenic plants with the bathochromic phytochromes showed significantly increased shade tolerance compared to wild-type plants and transgenic plants with wild-type phytochromes. Therefore, the present invention can be utilized to suppress plants&#39; shade avoidance that is one of major causes to induce crop-yield losses, and ultimately to generate shade tolerant plants with higher yields. 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 shade tolerance.

Description:
BACKGROUND OF THE INVENTION  
       [0001]     1. Field of the Invention  
         [0002]     This invention relates to a modified phytochrome A (PHYA) nucleic acid molecule of which Pr absorption spectra have been shifted to longer wavelength (i.e. bathochromic or red-shifted phytochrome A), in order to give plants shade-tolerance that increases crop yields. The phytochrome A functions as the photoreceptor in far-red wavelength light in mediating the suppression of shade avoidance and the development of plants. These bathochromic phytochromes absorb and utilize light even in the canopy and shade conditions, which suppresses shade avoidance reactions in plants (i.e. shade tolerance). Since the shade avoidance reactions re-distributes energy and resources to unnecessary elongation and acceleration of senescence (i.e. early flowering), it causes drastic reduction in products such as leaves, storage organs and seeds. Thus, the developed bathochromic phytochromes enable us to develop shade tolerant plants with high yields. The present invention also provides the methods and processes for generating transgenic plants transformed with the said nucleic acid molecules to engineer shade tolerance of economically important crop plants for high-yielding.  
         [0003]     2. Description of Prior Art  
         [0004]     Light is the most important environmental factor for optimal growth and development of plants (Chen et al., 2004). They harness not only energy from light for anabolic pathways that construct their building blocks, but also adapt to changes in light during their life cycle from germination to flowering. Phytochrome is a photoreceptor that manage a variety of photomorphogenic responses to the red/far-red region of the spectrum (Smith, 2000). They are dimeric chromopeptides (monomer sizes of 120˜130 kDa) that carry the chromophore phytochromobilin (PΦB), which is covalently linked to a cysteine residue on each peptide via a thioether linkage. There are two spectrally distinct forms of phytochromes, a red-light (R, λmax=660 nm) absorbing Pr form and a far-red light (FR, λmax=730 nm) absorbing Pfr form ( FIG. 1A ). The latter form is considered as the active form of phytochrome because of the promotive effect of red-light on most physiological responses. Phytochrome signaling in plants is driven by phototransformation between the two forms (Kim et al., 2002).  
         [0005]     Competition for sunlight is one of the most important aspects in regulating plant development (Ballare, 1999). Plants grown under dense canopies or at high density (i.e. shaded conditions) perceive a decrease in the ratio of R to FR light (R:FR ratio). This change in light quality serves as a warning of competition, triggering a series of responses known collectively as the shade avoidance syndrome or shade avoidance reactions (Smith and Whitelam, 1997; Devlin et al., 2003). The reactions in plants induce a rapid and dramatic increase in the extension growth of stems and petioles at the expense of leaf growth, storage organ production, and reproductive development (Table 1). Prolonged shade causes dramatically accelerated flowering, reduced seed sets, and immature fruits. The shade avoidance reactions are mediated predominantly by phytochromes (Smith and Whitelam, 1997). Phytochromes respond to the R:FR ratio as an indicator of proximity to and shade from neighbors. Since blue and red lights are selectively absorbed by chlorophylls for photosynthesis, far-red light is relatively enriched in shaded conditions ( FIG. 1B ). Thus, shade is represented as a low R:FR ratio. The enriched far-red light signal is recognized by phytochromes as a change in photoequilibrium between Pr and Pfr. The photoequilibrium of phytochrome is represented by the ratio [Pfr]/[Ptot], where [Ptot]=[Pr]+[Pfr]. The lowered R:FR ratio induces a decrease in the Pfr form of phytochromes, which trigger the shade avoidance reactions in plants.  
                             TABLE 1                           Shade avoidance reactions in plants.                Physiological process   Response to shade                       Germination   Retardation           Extension growth   Acceleration           Leaf development   Retardation           Chloroplast development   Retardation           Branching   Retardation           Flowering   Acceleration           Storage organ deposition   Severe reduction                      
 
         [0006]     In monocultures of crop plants in close proximity, competition for light is an important factor in determining crop yields, because it induces shade avoidance reactions such as elongation of internodes and petioles, inhibition of leaf expansion and growth, retardation of chloroplast development, and early flowering. Since the shade avoidance reaction re-distributes energy and resources to unnecessary elongation and acceleration of senescence ( FIG. 2 ), it cause drastic reduction in products such as leaves, storage organs and seeds. The overexpression of phytochromes in crop plants has been used to overcome these losses from shade avoidance reactions (Robson et al., 1996; Robson and Smith, 1997). FR light is enriched in the shade (See  FIG. 1 ), so the R:FR ratio is decreased. Phytochrome B (phyB) perceives the low R:FR ratio and rapidly induces shade avoidance reactions (Robson et al., 1993). Phytochrome A (phyA) has an antagonistic function to phyB in shade avoidance reactions (Botto et al., 1996). Thus, the  Avena  (oat) phyA gene has been introduced into crop plants such as tobacco, tomato, potato and wheat (Boylan and Quail, 1989; Heyer et al., 1995; Robson et al., 1996; Sineshchekov et al., 2001; Shlumukov et al., 2001). When constitutively expressed, the  Avena  phyA increases shade tolerance, resulting in improvements of leaf expansion and growth without the expense of elongation growth. Consequently,  Avena  phyA-overexpressing tobaccos showed shortened stature in low R:FR and proximity-conditional dwarfism in dense culture. The harvest index of transgenic tobacco showed approximately 20% improvement in leaf product (Robson et al., 1996). Also, transgenic tomatoes, potatoes and wheat displayed suppression of shade avoidance with improved leaf expansion and growth, greening and increased harvest indices of storage organs or seeds (Boylan et al., 1991; Heyer et al., 1995; Shlumukov et al., 2001).  
         [0007]     The increase of shade tolerance by overexpression of phytochromes is limited because of the limitation of expression levels and also the degradation of phytochrome proteins upon light illumination. Theoretically, an increase in Pfr under low R:FR ratios could mediate shade tolerance in plants. Thus, spectral phytochrome mutants that absorb longer wavelengths (i.e. bathochromic or red-shifted mutants in Pr absorption maxima) can be used to confer shade tolerance on plants. As shown in  FIG. 3 , the simulated spectra of Pr in bathochromic mutants shows an increase in the area overlapping with the shade spectrum, in which phytochromes recognize shade like red light and can be transformed to more Pfr form. Consequently, a shift of photoequilibrium to Pfr would overcome the shade avoidance. The absorption spectra of the phytochromes overlapped with the shade spectrum and the red-shifted mutants had greater overlap than the wild-type or blue-shifted mutant ( FIG. 3 ). The overlap of the 8 nm and 12 nm red-shifted mutants increased to 153±4% and 176±4% respectively, whereas the 8 nm blue-shifted mutant decreased to 67±4%. These calculations suggest that the red-shifted mutant can absorb more light in the shade, and maintain an increased amount of biologically active Pfr compared to wild-type phyA, thus conferring shade tolerance to plants. Thus, bathochromic phytochromes in this invention can be practically applied to suppress shade avoidance reactions under shaded conditions such as canopy and proximity cultures, which increase shade tolerance to plants with high yields. The 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  
       [0008]     The present invention relates to nucleic acid molecules encoding modified phytochrome A (phyA) protein of which Pr absorption spectra have been shifted to longer wavelength (i.e. bathochromism or red-shift). Such nucleic acid molecules confer shade tolerance to plants. Since the shade avoidance is second only to disease as a cause of crop-yield losses, the suppression of shade avoidance can significantly increase the crop yields. In this invention, several bathochromic phytochromes were developed and characterized photochemically and their biological functions were demonstrated by using transgenic  Arabidopsis  plants. The developed bathochromic phytochromes confer shade tolerance to plants: 6 nm bathochromic mutant (Red-6) had an increased shade tolerance of approximately 220% and 8 nm bathochromic mutant (Red-8) increased to approximately 270%, whereas 8 nm hypsochromic mutant (Blue-8) displayed a decreased shade tolerance. There is a proportional relationship between shade tolerance and wavelength shift in the absorption spectrum of the phytochrome. This means that the magnitude of the red shift is strongly correlated to level of shade tolerance. In this invention, 12 nm bathochromic mutant was developed. Therefore, this invention relates the development of bathochromic phytochromes and their application to develop shade-tolerant plants with high-yields. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0009]      FIG. 1  shows absorption spectra of phytochromes (A) and daylight spectra in open and canopy covered areas (B). In the canopy, only far-red light is enriched, which induces shade avoidance reactions in plants. [from Smith, 2000] 
         [0010]      FIG. 2  shows the phenotypes of  Arabidopsis  plants caused by shade avoidance. In high R:FR ratios, the relative [Pfr] is increased and shade avoidance responses are suppressed, including inhibition of stem elongation and flowering. A reduced R:FR ratio causes photoconversion from Pfr to Pr and the relative [Pfr] is decreased, which induces shade avoidance reactions.  
         [0011]      FIG. 3  shows simulated spectra of Pr in 8 nm blue-shifted (Blue-8), 8 nm red-shifted (Red-8), and 12 nm red-shifted (Red-12) spectral mutants and overlaps of these spectra with the shade spectrum of the LED growth chamber. Each red-shifted mutant shows an increase in area of the spectrum which overlaps with shade. The changes in area of overlap were calculated relative to the overlap between wild-type phyA (phyA) and shade (100%). The 8 nm red-shifted phyA overlap was 153±4%, whereas the 8 nm blue-shifted phyA overlap was 67±4%. The 12 nm red-shifted phyA showed greater increase than the 8 nm-shifted mutant (176±4%). These values can also reflect the changes in photoequilibrium.  
         [0012]      FIG. 4  shows difference spectra of wavelength-shifted mutant phytochromes assembled with PΦB. (A) with 8 nm blue-shifted (R317E), 6 nm red-shifted (F389A), and 8 nm red-shifted (F307R/C371A) mutants. (B) with 12 nm red-shifted mutant (F307R/Y385A/F389A) that showed maximally red-shifted absorption spectrum. Wt-PhyA, wild-type recombinant phyA. Gray lines show Pr peaks.  
         [0013]      FIG. 5  shows the effects of wavelength-shifted phytochromes on hypocotyl elongation in seedlings under shade. Around 30 individual 6-day-old transgenic and control seedlings were used to measure hypocotyl elongation. SD, short day (8 hrs: 16 hrs=light: dark); EOD-FR, end of day far-red light (15 min treatment of FR, 10 μmole/m 2 ·s). (A) Representative phenotypes in control and transgenic seedlings under SD or SD+EOD-FR condition. (B) Relative sensitivity of wavelength-shifted phyA in seedlings in the shade, as indicated by hypocotyl lengths in seedlings grown under SD+EOD-FR. Sensitivity (%)=(average of hypocotyl lengths of each plant/average of hypocotyl length of Col-0)×100.  
         [0014]      FIG. 6  shows representative phenotypes (A) and comparison of shade tolerance (B) of adult transgenic plants incubated in shade conditions. LD, long day (16 hrs: 8 hrs=light: dark); LD+FR2, W:FR=60 μmole/m 2 ·s:10 μmole/m 2 ·s; W, white light. wt-OX, oat phyA-overexpressing transgenic plant; Blue-8, 8 nm blue-shifted R317E transgenic plant; Red-6, 6 nm red-shifted F398A transgenic plant; Red-8, 8 nm red-shifted F307R/C371A transgenic plant. Relative shade tolerance (%)={(ratio of leaf area/petiole length of each transgenic plant)/(ratio of leaf area/petiole length of Col-0)}×100.  
         [0015]      FIG. 7  shows the effect of bathochromic phytochromes on proximity responses (A) and the comparison of leaf area under close proximity conditions (B). In A, 40 transgenic or control plants were planted in 10 cm diameter pots and cultured in LD for 6 weeks. wt-OX, oat phyA; Blue-8, R317E; Red-6, F398A; Red-8, F307R/C371A. In B, transgenic and control plants were planted at densities of 6 or 40 plants per 10 cm diameter pots and cultured in LD for 6 weeks. 12 transgenic or control plants were measured. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0016]     Phytochromes are dimeric chromopeptides (monomer sizes of 120˜130 kDa) that carry the chromophore phytochromobilin (PΦB), which is covalently linked to a cysteine residue on each peptide via a thioether linkage.  Avena  (oat) phyA consists of 1129 amino acids and the chromophore is attached at Cysteine 322 (Cys322 or C322). To generate absorption wavelength-shifted phyA mutants, bathochromic (shifted to longer wavelength, red-shifted) or hypsochromic (shifted to shorter wavelength, blue-shifted), the interactions between the chromophore and the surrounding amino acid residues in the chromophore-binding region were modified by site-directed mutagenesis. Approximately 30 candidate sites including 18 ring-bearing amino acids were selected and changed to another amino acid by site-directed mutagenesis. The full-length mutant phytochromes were expressed in the  Pichia  protein expression system, assembled with chromophore, phytochromobilin (PΦB), and their photochemical properties were analysed by using spectrophotometers. From these analyses, several absorption wavelength-shifted mutants were obtained (Table 2): two 2 nm hypsochromic, one 8 nm hypsochromic, three 2 nm bathochromic, four 4 nm bathochromic, six 6 nm bathochromic, two 8 nm bathochromic, one 10 nm bathochromic, and one 12 nm bathochromic mutants (total 3 hypsochromic and 17 bathochromic mutants).  
                                                   TABLE 2                           Absorption wavelength-shifted mutants in this invention.            Photochemical   Spectral               characteristics   change   Mutation   Remark                    Bathochromic   +2   D308K (SEQ ID NO: 11)   Red-       shift   +4   Y263F (SEQ ID NO: 12)   shifted               F307W (SEQ ID NO: 13)               Y326F (SEQ ID NO: 14)           +6   Y385A (SEQ ID NO: 1)               Y385F (SEQ ID NO: 2)               Y385V (SEQ ID NO: 3)               Y385W (SEQ ID NO: 4)               F389A (SEQ ID NO: 5)               Y385W/F389A (SEQ ID NO: 6)           +8   F389Y (SEQ ID NO: 7)               F307R/C371A (SEQ ID NO: 8)           +10   F307R/C371A/Y385W/F389A               (SEQ ID NO: 9)           +12   F307W/Y385A/F389A               (SEQ ID NO: 10)       Hypsochromic   −2   S310V (SEQ ID NO: 15)   Blue-       shift   −8   R317E (SEQ ID NO: 16)   shifted                  
 
         [0017]     Several absorption wavelength-shifted mutant proteins were assembled with chromophores and purified for further analyses. The difference spectra of several mutants were shown in  FIG. 4 , including 8 nm hypsochromic R317E mutant, 6 nm bathochromic F389A mutant, 8 nm bathochromic F307R/C371A double mutant, and 12 nm bathochromic F307W/Y385A/F389A triple mutant. Thus, several bathochromic phytochromes were successfully developed by site directed mutagenesis in the chromophore-binding region.  
         [0018]     Phytochromes manage a variety of photomorphogenic responses to the red/far-red region of spectrum (R/FR). Particularly, seed germination and shade avoidance are unique R/FR light responses regulated by phytochromes (Botto et al., 1996; Smith and Whitelam, 1997; Ballare, 1999; Sullivan and Deng, 2003). Light responses by phytochromes showed a positive correlation with the concentration of Pfr forms, [Pfr]. The initiation of shade avoidance reactions is determined by a dynamic photoequilibrium of phytochrome, and the photoequilibrium of phytochrome is represented by the ratio [Pfr]/[Ptot], where [Ptot]=[Pr]+[Pfr]. Even though enough photosynthetically active radiation may exist in the surroundings of a plant, the enriched and reflected FR from neighbors or leaves lowers the ratio of R:FR (See  FIG. 1 ). The lowered R:FR ratio induces a decrease in the Pfr form of phytochromes. Thus, bathochromic phytochromes in which Pr absorption spectra have been shifted to longer wavelength can absorb more light even in the low R:FR, which suppresses shade avoidance in plants. In this invention, several wavelength-shifted phyA mutants were selected and transformed into phyA-deficient  Arabidopsis thaliana  (phyA-211) to investigate the in vivo function of the mutants, especially under shaded conditions. Among the wavelength-shifted mutants listed in Table 2, R317E was selected as the representative blue-shifted mutant (8 nm), and F389A and F307R/C371A as representative red-shifted mutants, 6 nm and 8 nm shifts, respectively. Phenotypic characterization of the transgenic plants was performed to identify features positively correlated with the change in absorption spectra and the tolerance of shade avoidance. In addition, since the shade avoidance is second only to disease as a cause of crop-yield losses, the suppression of shade avoidance (i.e. shade tolerance) by using red-shifted phytochromes will have potential for biotechnological applications, for example increase of crop yields by suppressing shade avoidance, and increase in the value of decorative plants such as turfgrass.  
         [0019]     Shade avoidance reactions in plants induced by low R:FR ratios (i.e. shade) stimulate petiole elongation, retardation of leaf expansion, and floral induction (Morelli and Ruberti, 2000; Delvin et al., 2003). Additionally, hypocotyl elongation is very sensitive to light conditions: therefore the hypocotyl lengths of transgenic plants were tested. In these experiments, the R:FR ratio was modified by adding a far-red light period at the end of the day (EOD-FR) or by mixing the far-red light with white light irradiance (far-red light supplemented white light). To compare shade tolerance in seedlings, wild-type and transgenic seedlings were grown in 16-h light/8-h dark (long-day, LD) cycles for 3 days prior to growth for 3 days under 8-h light/16-h dark (short-day, SD) cycles, with or without a 15 min EOD-FR treatment. The wt-OX seedlings showed shorter hypocotyl lengths than non-transgenic wild-type seedlings (Col-0) with the EOD-FR treatment ( FIG. 5A ). The Blue-8 transgenic seedlings showed a hypersensitive phenotype to shade, compared to Col-0. The two transgenic seedlings with red-shifted phyA displayed less hypocotyl elongation than Col-0 and wt-OX. Red-8 plants reduced the shade sensitivity approximately 35% compared to Col-0 ( FIG. 5B ). In comparison, Blue-8 plants displayed approximately 10% increased shade sensitivity compared to Col-0. These results suggested that the red-shifted phyA could confer shade tolerance in seedlings, while the blue-shifted mutant confer shade sensitivity.  
         [0020]     To investigate the effects of bathochromic phytochromes in the shade avoidance responses in adult plants, transgenic plants were grown in a long day (LD) cycle for two weeks and transferred to white light with supplementary far-red light conditions in a LED (light emitting diode) growth chamber ( FIG. 6A ). Since shade avoidance responses are represented as complex changes in phenotypes including elongation of petioles and hypocotyls, retardation of leaf growth, and floral induction in  Arabidopsis , these phenotypes were investigated with the transgenic plants of wavelength-shifted phyA mutants. The ratio of leaf area to petiole length is influenced by light quality. In the shade, leaves are smaller and petioles are longer than under high R:FR. Therefore, changes in the ratio of leaf area/petiole length can be a measure of shade tolerance. Relative shade tolerance was calculated as shade tolerance (%)={(leaf area/petiole length of transgenic plant)/(leaf area/petiole length of Col-0)}×100. Wt-OX plants showed 150% increase in shade tolerance ( FIG. 6B ), reflecting the increased protein stability of monocot ( Avena ) phyA in dicot ( Arabidopsis ) plants, as well as higher expression levels of phyA under the constitutive promoter. Red-6 had an increased shade tolerance of approximately 220% and Red-8 increased to approximately 270%, whereas Blue-8 displayed a decreased shade tolerance (i.e. hypersensitive to shade). These data indicate that there is a proportional relationship between shade tolerance and wavelength shift in the absorption spectrum of the phytochrome in leaf morphology.  
         [0021]     In the shade, most blue and red light is reflected or absorbed by leaves and far-red light is enriched. Under these conditions, phyA accelerates floral induction by inhibiting the phyB-mediated suppression of flowering (Hayama and Coupland, 2003; Cérdan and Chory, 2003). Therefore, it is predicted that transgenic plants with red-shifted phyA would delay the shade-induced flowering. The results indicate that the red-shifted mutants displayed late floral induction in low R:FR (supplementary far-red light), compared to Col-0, whereas the blue-shifted mutant had little affect on floral induction (Table 3). Thus, the red-shifted phytochromes induced a delay in floral induction, supporting that phyA recognizes the low R:FR (i.e. shifts the photoequilibrium of Pr:Pfr) and induces flowering. The amount of Pfr in the transgenic of red-shifted mutants is relatively higher than that in the transgenic of wt-OX or Col-0, and this delayed initiation of flowering in red-shifted mutants, whereas initiation of flowering was slightly accelerated in the blue-shifted mutant.  
                                               TABLE 3                           Effect of wavelength-shifted phytochromes on flowering in shade                White   White + FR                            Col-0   9.83 ± 0.83   6.42 ± 0.49           Wt-OX   9.50 ± 0.50   6.82 ± 0.37           Blue-8   9.08 ± 0.75   6.25 ± 0.72           Red-6   9.92 ± 0.49   7.92 ± 0.64           Red-8   10.2 ± 0.68   8.83 ± 0.37                         Data are number of rosette leaves at bolting. White light (60 μmole/m 2 ), FR (10 μmole/m 2  · sec).                They were cultured in plastic boxes to maintain humidity in the LED growth chamber.             
 
         [0022]     In shade avoidance responses, the plants perceive the relative amounts of red light and undergo changes in photoequilibrium with enriched far-red light (Neff et al., 2000; Gilbert et al., 2001). Shorter wavelengths of light are scattered or absorbed by leaves and longer wavelengths (far-red) are reflected by neighbors and enriched in dense areas, such as deep forests. To compete for light, plants monitor their neighbors by analyzing the R:FR ratio by phytochromes. To measure the response of wavelength-shifted mutants to culture density, synchronized plants at the same developmental stage were transferred to a 10 cm-diameter pot. High-density cultures consisted of 40 plants per pot and control cultures contained 6 plants per pot. All plants were cultured in white light on a long day cycle. In high density cultures, Col-0, wt-OX and Blue-8 plants showed retardation of growth and induced flowering, whereas red-shifted mutants showed a delay in flowering and relatively increased leaf area ( FIG. 7A  &amp; Table 4). High-density cultures of Red-8 showed over 20% increase in leaf area compared to Col-0, and Blue-8 displayed reduced leaf area compared to Col-0 ( FIG. 7B ). Col-0 leaf area was similar tendency to the results of the shade experiment. However, the stress of close proximity did not show drastic changes in leaf morphology, compared to shade conditions. Red-shifted mutants in the high-density cultures delayed flowering by approximately two pairs of rosette leaves, compared to Col-0. Red-shifted mutants in the high-density cultures showed almost same flowering time as in the low-density cultures, indicating that the bathochromic phytochromes conferred the tolerance to the floral initiation by the high-density culture.  
                                               TABLE 4                           Effect of wavelength-shifted phytochrome on flowering in proximity.                6 plants/pot   40 plants/pot                            Col-0   11.83 ± 0.68    8.04 ± 0.76           wt-OX    11.5 ± 0.5     9.68 ± 1.06           Blue-8   10.48 ± 0.75    8.64 ± 0.98           Red-6   11.92 ± 0.49   10.14 ± 1.06           Red-8    13.2 ± 0.68   12.78 ± 0.8                         Data are number of rosette leaves at bolting.             
 
         [0023]     Overexpression of phytochromes, especially the transformation of monocot phyA into dicot plants, has been used for suppression of shade avoidance reactions, eventually increasing the harvest yields (Robson et al., 1996; Robson and Smith, 1997). The oat phyA gene has been introduced into crop plants such as tobacco, tomato, potato and wheat (Boylan and Quail, 1989; Heyer et al., 1995; Robson et al, 1996; Sineshchekov et al., 2001; Shlumukov et al., 2001). In transgenic tobacco overexpressing oat phyA, the harvest index was increased about 20% (Robson et al., 1996), and transgenic potatoes overexpressing phyB had increased tuber yield and decreased the density effects (Boccalandro et al., 2003). In most transgenic plants, including tomato, potato, and wheat, the overexpression of phytochromes suppresses shade avoidance and promotes improved leaf expansion and growth, greening, and increased harvest index for storage organs or seeds. Therefore, the transgenic approach with phytochrome genes has potential for biotechnological applications, especially for the increase of crop-yields by reducing losses due to shade avoidance reactions. In this invention, bathochromic phytochromes have developed by site-directed mutagenesis and it has been demonstrated that transgenic plants with bathochromic phytochromes possess improved shade tolerance. Furthermore, the magnitude of the red shift was strongly correlated to level of shade tolerance: an 8 nm red-shifted mutant showed more shade tolerance than a 6 nm red-shifted mutant (270% vs. 220% compared to Col-0). This means that the combination mutants with a 10-12 nm red shift can suppress the shade avoidance reactions more strongly than mutants with a 6-8 nm shift. These results suggest that the biological function of phytochromes is directly related to its photochemical properties and the more red-shifted a phyA mutant, the greater the shade tolerance it confers. In comparison, the blue-shifted mutant resulted in increased shade sensitivity. The results in this invention demonstrate that altered phytochromes will be an important tool for plant biotechnology. Since shade avoidance is one of major causes to induce crop-yield losses, introducing the red-shifted phytochromes into crop plants could prevent substantial losses in crop yields. Furthermore, decorative plants such as turfgrass could be improved. The transgenic turfgrass would be shorter and greener in the shade, thus increasing the decorative value. Therefore, this invention enables us to develop shade tolerant plants with high yields and values.  
       EXAMPLES  
       [0024]     All chemical reagents used were purchased from Sigma (St. Louis, Mo.) unless specified otherwise. Restriction and modifying enzymes were obtained from New England Biolabs, Inc. (Beverly, Mass.) and Roche Molecular Biochemicals (Indianapolis, Ind.). GeneEditor™ in vitro Site-Directed Mutagenesis System was purchased from Promega (Madison, Wis.). All polymerase chain reactions (PCR) were performed using high fidelity DNA polymerase, Turbo® Pfu polymerase which was purchased from Stratagene (La Jolla, Calif.). For the expression of all recombinant phytochromes, the  Pichia pastoris  protein expression system from Invitrogen (Carlsbad, Calif.) was used.  
         [0000]     Generation of Wavelength-Shifted phyA Mutants by Site-Directed Mutagenesis  
         [0025]     To perform site-directed mutagenesis in  Avena  (oat) phyA, a full-length phytochrome gene was subcloned into pGEM-11zf (+) (Promega, Medison, Wis.) from pFY122 (Boylan and Quail, 1989) by digesting with BamHI and EcoRI. To create Kozak sequence ( accatgg ) in the construct for protein expression in  Pichia , PCR amplification was performed with a forward primer, 5′-cg ggatcc accatggcttcctcaaggcctgcttcc-3′ (underlined, BamHI) (SEQ ID NO: 17), and a reverse primer, 5′-cg cccggg ctgcagagc tagatatagcatc-3′ (underlined, SmaI) (SEQ ID NO: 18). The amplified PCR products were digested with BamHI and AvrII, and replaced with the corresponding fragments of wild-type phyA in pGEM-11zf (+). The clones were confirmed by DNA sequencing. The site-directed mutagenesis was performed by using either a GeneEditor™ in vitro site-directed mutagenesis system (Promega, Medison, Wis.) or a QuickChange™ kit (Stratagene, La Jolla, Calif.) according to the manufacturer&#39;s recommendations. Mutagenic primers with base substitutions used in this study were listed in Table 5. To be convenient for screening, a proper restriction site was generated into each mutagenic primer or removed from original restriction site. The mutagenized plasmids were confirmed by restriction and DNA sequencing.  
         [0000]     Phytochrome Constructs for Recombinant Protein Preparations  
         [0026]     Full-length oat phyA was first subcloned into the pASK75 vector to attach a streptavidin affinity tag (strep-tag) at the end of the gene as described (Kim et al., 2004). Prior to the subcloning of phyA gene, the pASK75 vector was modified to have a NotI restriction site at the end of the strep-tag for further subcloning into  Pichia  expression vector, pPIC3.5K (Invitrogen, Carlsbad, Calif.). The strep-tag is composed of ten amino acids and strongly binds to streptavidin protein, which is used for affinity chromatography with streptavidin agarose (Sigma, St. Louis, Mo.). Specific primers for PCR amplification were designed to make an in frame construct with the strep-tag, 5′-cg ggatcc accatggcttcctcaaggcctgcttcc-3′ (forward, BamHI) (SEQ ID NO: 17) and 5′-tcgc gtcgac ttgtcccattgctgttggagc-3′ (reverse, SalI) (SEQ ID NO: 19). After confirmation of the constructs by restriction map analysis and DNA sequencing in pASK75, the phyA gene was subcloned into the pPIC3.5K vector using BamHI and NotI for the protein expression in  Pichia pastoris . To subclone mutant phytochromes, 1.6 kb of fragments (BamHI and AvrII) in wild-type phyA gene were exchanged with the fragments of each phyA mutant digested by the same restriction enzymes. After the phyA mutant genes were subcloned into pPIC3.5K, they were all confirmed by DNA restriction map analysis and DNA sequencing.  
                                 TABLE 5                           Mutagenic primers used for this invention.            Mutagenic       Restriction           site   Mutants   enzyme   Mutagenic primer sequence               Y263   Y263F   Stu I (X)   ggtcttgagccttttcttggactgcactatcc (SEQ ID NO: 20)       S296   S296A   BssH II   gattgccgt gcgcgc gccataaaaggtc (SEQ ID NO: 21)       F307   F307R   Sac II   gaggcactc ccgcgg gatattagcctatg (SEQ ID NO: 22)           F307W   Nco I   gctgaggcactc ccatgg gatattagcctatgtgg (SEQ ID NO: 23)       D308   D308K   DraI   gcactcccc tttaaa atttgcctatgtg (SEQ ID NO: 24)       S310   S310V   EcoRV   ctccccttt gatatc gtcctatgtggttcag (SEQ ID NO: 25)       S314   S314L   Nhe I   gcctatgtgg gctagc actcagggcac (SEQ ID NO: 26)       R317   R317E   XhoI   ggttcagca ctcgag gcaccacacag (SEQ ID NO: 27)       Y326   Y326F   Pst I   cagttgtcac ctgcag tttatggagaacatg (SEQ ID NO: 28)       W366   W366F   N.A.   gaagaaactattcggcctccttg (SEQ ID NO: 29)       C371   C371A   AviII   ggcctccttgt tgcgca ccatgagagc (SEQ ID NO: 30)       Y385   Y385A   Sph I   ccgctgcgtgct gcatgc gagttcttagcacag (SEQ ID NO: 31)           Y385F   Sph I   ccgctgcgtttt gcatgc gagttcttagcac (SEQ ID NO: 32)           Y385V   Sph I   ccgctgcgtgttg gcatgc gagttcttagcacag (SEQ ID NO: 33)           Y385W   Sph I   ccgctgcgttgg gcatgc gagttcttagcacag (SEQ ID NO: 34)       F389   F389A   Stu I   gttatgcttgtg aggcctt agcacaggtg (SEQ ID NO: 35)           F389Y   Sca I   gttatgcttgtg agtact tagcacagg (SEQ ID NO: 36)       F307/C371   F307R/C371A   SacII/AviII   gaggcactc ccgcgg gatattagcctatg (SEQ ID NO: 37)                   ggcctccttgt tgcgca ccatgagagc (SEQ ID NO: 38)                 Cf. N.A., not applicable;            bold, created restriction enzyme sites;            (X), abolished original restriction site             
 
 Isolation and Purification of Chromophores 
 
         [0027]     Phytochromobilin (PΦB) and phycocyanobilin (PCB) were used as the chromophores for holo-phytochrome assembly in this work. PΦB was extracted from red algae,  Porphyridium cruentum  by methanolysis and subsequently purified by chromatography as followed previous report (Beale and Cornejo, 1991).  Porphyridium cruentum  cells were grown in minimal liquid medium at 27° C. under cool white and red fluorescent lights (1:1 ratio). The culture medium was aerated by magnetic stirring and continuous flushing with an air/CO 2  gas mixture. The harvested cells were washed with acetone until the supernatant was colorless. Then, the pellet was resuspended with 1 mg of HgCl 2  in 1 ml of the absolute methanol and incubated in darkness for 16-24 hours at 40° C. After methanolysis, the supernatant was applied to a C-18 Sep-Pak column (Waters—Millipores, Mass.), and washed with 0.1% trifloric acid (TFA) in distilled water. PΦB was fractionated with acetonitrile/0.1% TFA (60:40, v/v). The fraction containing PΦB can be detected at 370 nm. Phycocyanobilin (PCB) was purified from lyophilized  Spirulina platensis  powder purchased from Sigma. The lyophilized powder was resuspended into water and centrifuged to get supernatant. The supernatant was mixed with trichloroacetic acid (1%, w/v), and followed the methanolysis. It can be detected at 370 nm. The concentration of PCB and PΦB was determined by absorption spectroscopy in HCl (2%)/methanol, using extinction coefficients (ε) of 37,900 M −1  cm −1  at 690 nm for PCB and 64,600 M −1  cm −1  at 708 nm for PΦB, respectively. The purified pigments were dissolved in dimethyl sulfoxide (DMSO), wrapped with foil and stored at −80° C. until use.  
         [0000]     Expression and Purification of Mutant Phytochromes  
         [0028]     The pPIC3.5K constructs with the mutated phytochrome genes were transformed into  Pichia pastoris  GS 115 cells by electrophoration method, as previously described (Kim et al., 2004). This expression vector has two selective markers, histidine auxotrophic marker and geneticin (G-418) resistant marker. First, the transformants were selected on minimal media containing dextrose (MD, 0.34% yeast nitrogen base, 4×10 −5 % d-biotin, 2% dextrose) media plates to remove non-transformants. Then, the selected colonies from MD medium were spread out on yeast extract-peptone-dextrose (YPD, 1% yeast extract, 2% bactopeptone, 2% dextrose) agar plates containing 3 mg/mL of geneticin antibiotics to select out the  Pichia  transformants bearing multi-copy integrated phytochrome expression cassettes in the genomic DNA for protein expression. For the induction of proteins, the selected cells were grown on 5 mL of minimal medium containing methanol (MM, 1.34% yeast nitrogen base, 4×10 −5 % d-biotin, 1% methanol) at 30° C. with shaking at 250 rpm overnight, transferred to 100 mL of minimal medium containing glycerol (MGY, 1.34% yeast nitrogen base, 4×10 −5 % d-biotin, 1% glycerol) and cultured one more day until it reached 5.0 of optical density at 600 nm. The grown cells were harvested and transferred to 500 mL of MM media in 2 L of a baffled flask for the protein induction (the optical density to 0.8˜1.0). Then, the cells were cultured for 20-24 hours at 30° C. with shaking at 250 rpm for the protein induction. Cultured cells were harvested by centrifugation (4500 rpm, 5 min at 4° C.) and washed with 50 mL of sterilized water. The washed cell pellets were resuspended with 10 ml of TE buffer (100 mM Tris-HCl, pH 8.0, 1 mM EDTA) containing protease inhibitors, including 1 mM phenylmethyl-sulfonyl fluoride (PMSF), 4 μg/mL leupeptin and 4 μg/mL pepstatin. Resuspended  Pichia  cells were homogenized in liquid nitrogen with a homogenizer (Nihonseiki Kaisha, Japan; model AM-5) for 5 min at 13,000 rpm twice. The disrupted cell extracts were centrifuged at 15,000 rpm for 20 min under 4° C. The supernatants containing the apo-phytochromes were precipitated with ammonium sulfate (0.23 g/ml) to fractionate from cell extract contaminants. The re-solublized precipitants in TE buffer were directly used for in vitro chromophores-adduct. The purified chromophores, PCB or PΦB was added to each supernatant at a final concentration of 20 μM and stood on ice for 1 hr under the dark. For the protein purification, chromophore-adducted samples were dialyzed for 2 hrs under dark at 4° C. to remove excessive salts from the samples. The dialyzed samples were passed through a 0.45 μm microfilter (Nalgene) to remove any insoluble particles and loaded onto streptavidin-affinity column for purification. The column was washed with TE buffer until optical density at 280 nm was dropped to under 0.01 and eluted the recombinant proteins with 5 mM desthio-biotin containing TE buffer.  
         [0000]     Qualitative and Quantitative Analyses of Purified Phytochromes  
         [0029]     Protein samples were analyzed by SDS-PAGE using 10% polyacrylamide minigels and were stained with 0.25% Coomassie Brilliant Blue R250. For Western blot analysis, the protein bands on the SDS-PAGE gel were transferred to a PVDF membrane (Hybond-P, Amersham-Pharmacia), and the membrane was incubated with oat phytochrome A-specific monoclonal antibodies, oat-22 and oat-25 (Cordonnier, 1989), for 2 hours, and developed by using an ECL™ western blotting analysis system (Amersham). To investigate whether the chromophore ligated with phytochrome proteins, Zinc blot analysis was carried out as described (Berkelman and Lagarias, 1986). The protein samples were separated on a SDS-PAGE gel and soaked in 20 mM zinc acetate/150 mM Tris-HCl, pH 7.0 for 5-30 min at room temperature with gentle agitation. The chromophores covalently linked phytochrome was visualized under UV light (312 nm) as a bright pink colored band. The concentrations of protein samples were determined by Bradford method using bovine serum albumin (BSA) as a standard.  
         [0000]     Photochemical Analyses of Recombinant Phytochromes  
         [0030]     All experiments were performed under safety green light conditions with a maximal transmittance at 500 nm through a specific filter (Rosco). The absorption spectra of holo-phytochromes were recorded in the range between 260 and 800 nm by a diode array UV/VIS spectrophotometer (Varian, Cary3 Bio EL97063574). The absorption spectra of the Pr and Pfr forms of each mutant phytochrome were measured after red or far-red light irradiation. A fiber optic illuminator system (Cole-Palmer) equipped with 656 and 730 nm interference filters (Oriel) was used as a light source. The light intensity was 8 W/m 2  for red light and 6 W/m 2  for far-red light. Red or far-red lights were illuminated to each sample for at least 2 min. A difference spectrum was calculated by subtracting the Pr spectrum from the Pfr spectrum, or reverse subtraction. From the absorption and difference spectra, the absorption maxima of Pr and Pfr (λ pr  and λ pfr ) were determined. To investigate non-photochemical reversion of Pfr to Pr (dark reversion), phytochromes were irradiated with red light to transform Pfr. The amounts of [Pfr] and [Ptot] were then checked in a time-dependant manner with UV/VIS spectrophotometer at room temperature (Varian, Cary3 Bio EL97063574).  
         [0000]     Construction of Plant Expression Vectors  
         [0031]     Oat phyA gene and wavelength-shifted mutant genes were subcloned into pCambia 1200 binary plasmid containing a hygromycin selective marker. For expression of phytochromes, CaMV (Cauliflower mosaic virus) 35S promoter and NOS terminator originated from the nopaline synthase gene of  Agrobacterium  were used in all plant expression constructs. The fragment of oat phyA gene was prepared with sequential enzymatic treatment: EcoRI digestion, T4 polymerase treatment for making blunt end at 3′ end prior to BamHI digestion for 5′ end. This fragment was ligated with the digested vector using the BamHI and EcoICRI. The pCambia1200 containing oat phyA gene was then used for subcloning of the wavelength-shifted mutant phytochromes. As exchanging each fragment from KpnI to AvrII between the wild type and wavelength-shifted mutants, the mutated phytochrome genes were easily subcloned into plant expression vector.  
         [0000]     Plant Growth Conditions and Transformation  
         [0032]      Arabidopsis thaliana  ecotype Col-0 and phyA mutant allele, phyA-211 provided from  Arabidopsis  Biological Research Center at Ohio (ABRC), were used in all experiments (Reed et al, 1994). The plant expression vectors containing each wavelength-shifted mutant and wild type were transformed into  Agrobacterium tumefaciens  GV3101. With each transformed  Agrobacterium, Arabidopsis  transformation was followed by  Agrobacterium -mediated floral dip method (Clough and Bent, 1998). For the transformation,  Arabidopsis  plants were grown to flowering stage at 22-24° C. at long day condition. The transformed  Agrobacterium  was grown at 28° C. in sterilized YEP (10 g tryptone, 10 g yeast extract, 5 g NaCl per liter of water) including 50 μg/mL of hygromycin. The cultured transformants were harvested and resuspended in infiltration medium to approximately 0.8 of a final OD 600  prior to use. Infiltration medium is composed of ½ strength MS basal medium, 5.0% sucrose, 0.44 μM benzylamino purine, 0.005% Silwet L-77 and B5 vitamins. After the transformation by floral dip method, the plants were cultured under cool white light at 22-24° C.  
         [0000]     Selection and Analyses of Transgenic Plants  
         [0033]     After transformation, seeds (heterozygous T1 line) were harvested and surface sterilized as follows: treated with 95% ethanol for 30-60 sec, then with 10% (v/v) commercial bleach containing 0.05% Tween-20 for 5 min, followed by three times rinses with sterile water. The washed seeds were stored at cold and dark room for 3˜5 days to synchronize the germination. To obtain transgenic plants of homozygous wild type (wt-OX) and each wavelength-shifted mutants, the sterilized seeds were sown on hygromycin-selection plates containing 0.5×MS medium, 0.8% phytoagar and 50 μg/mL of hygromycin. Transgenic plants were identified as antibiotics-resistant seedlings that produced green leaves and well-established roots on the selective medium. The grown plants on the selective media were allowed to self-pollinate by transplanting into heavily moistened potting soil. The harvested seeds (heterozygous T2 line) from the first screening were tested again to obtain 3:1 segregated plants, which contain single allele of T-DNA in their chromosomes. To obtain homozygous T3 line, the grown young plants tested from 3:1 segregation on plates containing antibiotics were transferred to moistened soil. Then, the seeds from each transformed plant (T3 seeds) were sown on plats containing antibiotics and selected as homozygous line when all of the sown seeds were grown up to show antibiotics resistance. The screened homozygous transgenic lines were confirmed by Western blot, RT-PCR (Reverse Transcription-Polymerase Chain Reaction), and genomic southern blot analysis. All physiological experiments were performed with the T3 seeds.  
         [0000]     Light Sources and Spectral Measurements  
         [0034]     Plants were illuminated with fluorescent cool white light for the growth. Monochromic far-red light (photon irradiance approximately 700˜800 nm, peak of maximum 738 nm) was provided from a light emitting diode (LED) array at LED incubator (VS-9108M-LED, Vision Scientific Co. Seoul, Korea). The used photon fluence rate was measured by radiometer IL-1700 (International Light, Newburyport, Mass.) with detector SED033 (#7963, International Light).  
         [0000]     Physiological Analyses  
         [0035]     To synchronize seed germination, sterilized seeds were kept in the dark at 4° C. for 3˜5 days, then exposed under white light for 1˜2 hrs, and kept in the dark for 1 day prior to the treatment for a specific light conditions in experiments (Fankhauser and Casal, 2004).  
         [0036]     To test shade sensitivity of the transgenic plants, wild-type and mutant phyA transgenic seedlings were grown in long-day cycles (LD, 16-h light/8-h dark cycle) for 3 days (white light, 60 μmole/m 2 ·s) followed grown in short-day cycles (SD, 8-h light/16-h dark cycle) for 3 days without or with a 15 min end-of-day far-red light treatment which mimics the shade condition (FR, 10 μmole/m 2 ·s) (Devlin et al., 1999). Also, to investigate the shade tolerance from adult plants, they were cultured under LD condition for 2 weeks prior to transferring to LED growth chamber to each different light condition (LD+FR1, W:FR=60 μmole/m 2 ·s:5 μmole/m 2 ·s; LD+FR2, W:FR=60 μmole/m 2 ·s: 10 μmole/m 2 ·s). Then, they were cultured for 3 weeks under different light conditions. The largest leaf from each transgenic plant was measured to investigate leaf areas and petioles length. The pictures of transgenic seedlings or plants were taken and the hypocotyls lengths, petiole lengths, leaf-lengths and leaf-widths were measured by using NIH image analyzer program.  
         [0037]     To investigate the effect of wavelength-shifted phytochromes on the recognition of proximity, 6 plants or 40 plants of each transgenic plant were planted in a pot with 10 cm diameter. To synchronize the developmental stage in the same pot, 2 week-grown young plants at the same stage were transferred to new pots.  
         [0038]     All the experiments were performed with each two different homozygous plants containing each wavelength-shifted phytochrome genes. All experiments were repeated at least three times.  
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