Patent Application: US-64110003-A

Abstract:
this invention is about the functionally hyperactive light signal related molecule , hfr1 - δn105 , of which the nucleic acids that encode n - terminal 105 amino acid residues were deleted . hfr1 as a bhlh transcription factor functions in a subset of phytochrome a signaling cascade and it was reported to be regulated negatively by cop1 . experiments with a hfr1 - δn105 overexpressing plant revealed that the deletion of n - terminal amino acids makes the hfr1 more active in photomorphogenic development such as germination and de - etiolation . in addition , the transgenic plants showed hypersensitive photo - responses in the inhibition of hypocotyl elongation , dependently on another positive element of light signaling , a bzip protein , hy5 . the end - of - day far - red light response and petiole elongation were suppressed in the hfr1 - δn105 overexpressing plants . these results suggest that n - terminal region of hfr1 negatively regulate hfr1 function and that hfr1 - δn105 is hyperactive .

Description:
phytochromes are the best - characterized photoreceptor that regulates 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 . in arabidopsiss , phytochrome apoproteins are encoded by a small gene family , phya - e ( sharrock and quail , 1989 ). mutational and transgenic approaches have revealed that individual phytochromes have overlapping but distinct functions ( reed et al ., 1994 ; quail et al ., 1995 ; furuya and schäfer , 1996 ; whitelam and devlin , 1997 ). in particular , phya is a primary photoreceptor for fr - high irradiance response ( hir ) and very low fluence response ( vlfr ), whereas phyb is a primary photoreceptor for r - hir and r - low fluence response ( lfr ). the hfr1 is revealed as a basic helix - loop - helix protein that is required in a subset of phytochrome a ( phya )- mediated photo - responses in arabidopsis ( fairchild et al ., 2000 ; fankhauser et al ., 2000 ; soh et al ., 2000 ). to further investigate the functions of hfr1 in light signaling , we stably introduced hfr1 into mutant hfr1 - 201 and wild type arabidopsis . the hypocotyl phenotype of the mutant hfr1 - 201 was complemented by the hfr1 overexpression ( fig1 and 2 a ). however , the wild type plant overexpressing hfr1 did not show any phenotypic alterations . it suggests that the full - length hfr1 is not a limiting factor for the photo - responses and the negative control mechanism may be involved . to test this possibility , we generated two modified hfr1 genes : hfr1 - δn105 , which lacks the dna sequence encoding 105 n - terminal end amino acids , and hfr1 - δc45 , which lacks the dna sequence encoding 45 c - terminal end amino acids . these two modified hfr1s were stably introduced into arabidopsis . while the transgenic plants overexpressing hfr1 - δc45 did not exhibit any differences in the photo - responses ( data not shown ), the transgenic plants overexpressing hfr1 - δn105 showed shortened hypocotyls under light and dark conditions ( fig2 a ). in addition , the hfr1 - δn105 transgenic plants displayed cotyledon opening / expansion and apical hook opening in the dark ( fig3 ). the phenotypic severity of the transgenic lines appeared to correlate with the expression level of hfr1 - δn105 transgene ( fig2 b ). but the hfr1 - δn105 ox - 36 line exhibited shortened hypocotyls in darkness , even though the expression level of the transgene was lower than that of the lines expressing full - length hfr1 . this result indicates that the exaggerated photo - responses of the hfr1 - δn105 transgenic lines may not be simply due to the higher expression level of the hfr1 - δn105 transgene . taken together , it is possible to suggest that the n - terminal region of hfr1 is negatively controlled and hfr1 - δn105 is hyperactive conferring photomorphogenic development even in the absence of light . phytochromes mediate the induction of germination by light . treatment with a pulse of fr light just after imbibition inhibits germination , whereas subsequent irradiation with r light results in phyb - mediated seed germination ( shinomura et al ., 1996 ). thus , the phyb mutant seeds do not germinate even after the treatment of r light . so far , except the photoreceptor itself , no mutations that mediate phytochrome - dependent seed germination have been identified . however , the seeds of hfr1 - δn105 transgenic lines germinated after fr light treatment . it implies that the seeds are able to undergo light - independent germination ( fig4 ). the intragenic suppressor of hfr1 - δn105 overexpressor ( sup1 ) could restore the altered germination phenotype of hfr1 - δn105 ox - 9 , further suggesting that the constitutive germination was due to the overexpression of hfr1 - δn105 . these results indicate that hfr1 - δn105 affects phytochrome signaling that leads to germination . to examine the photo - responses of the hfr1 - δn105 overexpressing plants in the inhibition of hypocotyl elongation in detail , we grew seedlings under various fluence rates of r or fr light . compared to wild type , hfr1 - δn105 transgenic plants were hypersensitive to both r and fr light ( fig5 a and b ). together with the phenotype showing shortened hypocotyls in darkness , these results imply that not only can hfr1 - δn105 activate photo - responses independently of light conditions , but it can also act synergistically with other light - signaling components to inhibit hypocotyl elongation in the presence of light . in some cases , enhanced phya signaling could lead to hypersensitivity to r light as well as fr light ( hoecker et al ., 1998 ; büche et al ., 2000 ). to determine which photoreceptor mediates the hypersensitive photo - response of hfr1 - δn105 transgenic plants , we constructed phyahfr1 - δn105 ox , and phybhfr1 - δn105 ox double mutants . as shown in fig6 a and b , the enhanced photo - responses of hfr1 - δn105 ox plants under fr light or r light were absent in plants on the phya mutant or phyb mutant backgrounds , respectively . thus , the enhanced photo - responses of hfr1 - δn105 transgenic plants under fr or r light required functional phya or phyb , respectively . to examine the dependence of hfr1 - δn105 on the downstream signaling components of phya for its enhanced photo - responses , we generated double mutants of hfr1 - δn105 transgenic lines and the mutants , jhy1 or jhy3 , which are well known upstream components in phya signaling . fhy1 and fhy3 have been shown to define a distinct signaling branch ( desnos et al ., 2001 ; okamoto et al ., 2001 ; wang and deng , 2002 ). our experiments showed that both fhy1 and fhy3 are necessary for the shortened hypocotyls exhibited by hfr1 - δn105 transgenic plants ( fig6 b ). we also investigated the relationship between hfr1 - δn105 and hy5 , a bzip transcription factor that plays a positive role in light signaling , in that hy5 and hfr1 additively inhibit hypocotyl elongation under fr light ( kim et al ., 2002b ). we found that the enhanced light responses of hfr1 - δn105 overexpressing plants were decreased on the hy5 mutant background . in contrast , the hy5 mutation did not affect the shortened hypocotyl phenotype of hfr1 - δn105 overexpressing plants in darkness . thus , the results suggest that hfr1 - δn105 may function cooperatively with hy5 to inhibit hypocotyl elongation in response to light . in the case of plants grown under white ( w ) light , stable phytochromes , primarily phyb , mediate various light - responses , such as hypocotyl / petiole elongation ( whitelam and devlin , 1997 ). to test whether hfr1 - δn105 affects the low fluence response , which is primarily regulated by phyb , we examined end of day ( eod )- fr light response . while wild type seedlings exhibited longer hypocotyls under eod - fr light conditions , as compared to control short day conditions , phyb mutant plants exhibit a constitutive eod - fr light response . the overexpression of hfr1 - δn105 significantly suppressed hypocotyl elongation in response to eod - fr light treatment ( fig7 a ). the results indicate that hfr1 - δn105 ox - 9 is less sensitive to inactive phytochromes that would be formed by fr light treatment at the eod . in adult plants , petiole elongation was inhibited in hfr1 - δn105 transgenic plants under both long - day and short - day conditions ( fig7 b ). this result indicates that hfr1 - δn105 enhanced a subset of phytochrome signaling pathways in response to w light , including those that regulate hypocotyl and petiole elongation . the seeds of wild type ( col ), phyb - 9 and phya - 211 mutants lines were obtained from arabidopsis biological resources center ( abrc ) ( columbus , ohio ). the fhy3 - 1 seed was kindly provided by dr . garry whitelam ( leicester university , uk ) and the hy5 - 221 seed was obtained from dr . xing - wang deng ( yale university , new haven , conn .). the fhy1 - 311 mutant was derived from our mutant screening with ems - mutagenized seeds and was shown to be a null mutation ( unpublished result ). all mutants used are from col background . light conditions used were same as previously described ( soh et al ., 2000 ). for measurement of hypocotyl lengths , seeds were surface sterilized for 5 min in commercial bleach and rinsed with sterile distilled water at least five times . seeds were then sown onto ms medium containing 0 . 8 % agar . after incubation at 4 ° c . for 3 days , the plates were placed in w light for 12 hours at 23 ° c . to improve germination and then transferred to the appropriate light conditions . data were collected from 40 % of the longest seedlings , to minimize variation in hypocotyl lengths among the seedlings as described previously ( soh et al ., 1998 ). germination tests were performed as described by shinomura et al . ( 1996 ). seeds were surface - sterilized and sown on aqueous medium containing 0 . 7 % agar . seeds were irradiated with fr light ( 21 w / cm 2 ) for 15 min and then kept in darkness with or without a single pulse of r light ( 33 w / cm 2 ) for 10 min . after 5 days , germination frequency was determined . 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 .). 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 . 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 ). seedlings were grown on ms - sucrose ( 2 %) medium for 4 . 5 days in darkness and then transferred to fr light for the indicated times before harvesting under dim - green light . total cellular rna was extracted from whole seedlings using the rneasy miniprep kit ( qiagen , valencia , calif .). rna gel blot analysis was performed as described ( soh et al ., 1998 ). full - length hfr1 was amplified by polymerase chain reaction ( pcr ) with hfr1 cdna ( soh et al ., 2000 ) using primers hfr1f4 - 2 , 5 ′- cga gaattc atgtcgaataatcaagctttc - 3 ′ ( seq id no : 3 ) and hfr1r8 , 5 ′- cctaatttg gaattc ttttctctc - 3 ′ ( seq id no : 4 ) and the mutant hfr1 ( hfr1 - δn105 ) lacking the n - terminal 105 amino acids was generated by pcr using primers , hfr1f5 , 5 ′- cga gaattc atgagaaacaaacatgag - 3 ′ ( seq id no : 5 ) and hfr1r8 . the ecori sites introduced are underlined and the atg start codon introduced for hfr1 - δn105 is shown in italic . the pcr products were digested with ecori and then cloned into binary vector pnb96 , obtained from dr . hong - gil nam ( postech , republic of korea ), in which transgene is driven 35 s dual promoter . the constructs were sequence verified . the resulting binary vector was introduced into agrobacterium gv3101 and used to transform wild type arabidopsis , col or hfr1 - 201 mutant . more than 40 independent t1 transgenic plants were selected . phenotypic analysis was performed with single t - dna insertion lines of at least 20 independent lines . to construct double mutants , we crossed hfr1 - δn105 transgenic plants with light - signaling mutants and allowed the f 1 progeny to self - pollinate to produce the f 2 seeds . basta - resistant plants were identified among the f2 seedlings and then grown for setting f3 seeds . the resulting f3 lines were tested for heterozygous basta - resistance and homozygous long - hypocotyl phenotypes under appropriate light conditions . from these , basta - resistant seedlings were selected and further grown to the f4 generation , and then plants were screened for homozygous basta - 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