Patent Application: US-12945905-A

Abstract:
the present invention includes modified phytochrome a nucleic acid molecules in which pr absorption spectra have been shifted to longer wavelength . 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 . 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 . 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:
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 ). several absorption wavelength - shifted mutant proteins were assembled with chromophores and purified for further analyses . the difference spectra of several mutants were shown in fig4 , 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 . 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 fig1 ). 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 . 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 ( fig5 a ). 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 ( fig5 b ). 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 . 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 ( fig6 a ). 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 ( fig6 b ), 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 . 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 . 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 ( fig7 a & 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 ( fig7 b ). 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 . 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 . 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 , a machine for preparing site - directed mutagenesis , was purchased from promega ( madison , wis .). all polymerase chain reactions ( pcr ) were performed using high fidelity dna polymerase , turbo ® pfu polymerase , a polymerase made by strategene , 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 . 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 barnhi and ecori . to create kozak sequence ( accatgg ) in the construct for protein expression in pichia , pcr amplification was performed with a forward primer , 5 ′- cgggatccaccatggcttcctcaaggcctgcttcc - 3 ′( underlined , bamhi ) ( seq id no : 17 ), and a reverse primer , 5 ′- cgcccgggctgcagagc tagatatagcatc - 3 ′( underlined , sinai ) ( 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 . ), a machine for preparing site - directed mutagenesis , or a quickchange ™ kit ( stratagene , la jolla , calif . ), a kit for preparing site - directed mutagenesis , 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 . 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 . 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 . the ppic3 . 5k constructs with the mutated phytochrome genes were transformed into pichia pastoris gs115 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 . 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 . 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 ). 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 . 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 . 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 . 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 ). 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 ). 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 . 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 . 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 . ballare , c . l . 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