Patent Application: US-35902699-A

Abstract:
a gene designated ckb3 whose product interacts specifically with cca1 has been identified through use of the yeast two - hybrid system . ckb3 is a structural and functional homologue of the regulatory subunit of protein kinase ck2 in arabidopsis . recombinant ck2 can phosphorylate cca1 in vitro . furthermore , arabidopsis plant extracts contain a ck2 - like activity that affects the formation of a dna - protein complex containing cca1 . recombinant plants that overexpress ckb3 have been constructed . overexpression of ckb3 results in increased ck2 activity and resulted in shorter periods of rhythmic expression of cca1 and lhy , as well as of four other circadian clock - controlled genes . this resulted a significant shortening of time to flowering under short - day conditions . this change in flowering time was not accompanied by significant phenotypic changes in morphology . alteration of ck2 activity , particularly through the overexpression of the ckb represents a new and effective way of modulating flowering time in plants .

Description:
the following description is provided to enable any person skilled in the art to make and use the invention and sets forth the best modes contemplated by the inventor of carrying out his invention . various modifications , however , will remain readily apparent to those skilled in the art , since the general principles of the present invention have been defined herein specifically to provide a new method of altering circadian rhythms and flowering in plants through the activation of ck2 , particularly by overexpression of β - subunits such as the newly - discovered ckb3 . standard methods of molecular biology were used in the experiments leading to the present invention . the basic methods are briefly listed below for the edification of one of skill in the art . saccharomyces cerevisiae y190 and pas2 , pas - snf1 , and pact were obtained from the arabidopsis biological resource center ( abrc ). ydh8 and pjcr14 have been described ( 20 , 45 ). pkt - ckb3 was constructed by ligating the arabidopsis ckb3 cdna into pkt10 ( 58 ). for the yeast two - hybrid screen , the entire coding region for cca1 was fused to the gal4 dna - binding domain ( gal4 - db ) in pas2 . y190 was transformed with the resulting plasmid , pas - cca1 , then with a library made from arabidopsis cdnas fused to the gal4 transactivation domain ( gal4 - ad ) ( abrc ). transformants ( 4 × 10 6 ) were analyzed as described previously ( 11 ). to obtain a cdna for the full - length ckb3 gene , an arabidopsis cdna library in λgt22 ( 63 ) was screened with the selected clone 106 . dna sequencing was done with a sequenase kit ( united states biochemical ). the genbank database was searched using the blast program . pgex - cca1 contains cdna encoding cca1 cloned into pgex - 3x ( pharmacia ). pet - cka1 , pet - cka2 have been described ( 39 ). pt7 - ckb1 and pt7 - ckb3 contain cdnas encoding arabidopsis ckb1 ( 10 ) and ckb3 in pt7 - his ( 50 ). expression and purification of glutathione - s - transferase ( gst ) and gst - cca1 and purification of cca1 by cleavage of gst - cca1 with factor xa have been described ( 63 ). cka1 and cka2 were produced as described ( 39 ), and were purified on a heparin - agarose column ( bio rad ). his - tagged ckb1 and ckb3 were produced in escherichia coli strain bl21 ( de3 ), and purified on ni - nta agarose ( qiagen ). protein concentrations were determined by the bradford assay ( bio - rad ). ck2 subunits and gbf4 labeled with [ 35 s ] methionine were synthesized by coupled transcription - translation with wheat germ extract ( promega tnt ). for in vitro binding , 20 μl of the reactions were added to 200 μl of binding buffer [ 20 mm hepes ph 7 . 6 , 100 mm kcl , 10 % glycerol , 5 mm edta , 0 . 02 % np40 , 1 mm dithiothreitol ( dtt ), 5 mg / ml bovine serum albumin ( bsa )] followed by 10 μl of glutathione - agarose beads with bound gst or gst - cca1 and incubated at 4 ° c . the beads were washed with binding buffer , then with binding buffer without bsa . bound proteins were eluted with 1x sds - polyacrylamide gel electrophoresis ( page ) sample buffer and resolved by 12 . 5 % sds - page . 35 s - labeled bands were detected by autoradiography , and quantitation was performed with a phosphorimager ( molecular dynamics ). gst - cca1 bound to glutathione - agarose beads was resuspended in 50 μl of ck2 buffer ( 50 mm tris - hcl , ph 7 . 5 , 10 mm mgcl 2 , 100 mm nacl , 1 mm dtt . 0 . 1 mm atp or gtp ) in the presence of 5 - 10 μci of [ γ - 32 p ] atp or gtp . the reaction was started by adding ck2 or whole - cell extracts ( wce ) and incubating samples at 30 ° c . for 30 min . wce were prepared as described previously ( 3 ) except that the phosphatase inhibitor cocktail ( 5 mm nh 4 vo 3 , 0 . 2 mm ammonium molybdate , 1 mm egta , 50 mm naf ) was added to extraction buffer . the beads were washed with phosphate - buffered saline ( pbs ) containing 1 % triton x - 100 and resuspended in 1x sds - page sample buffer . the phosphorylated samples were separated by 10 % sds - page . 32 p - labeled bands were detected by autoradiography and quantitated with a phosphorimager . cca1 was incubated with 0 . 1 ng of end - labeled a2 fragment of the arabidopsis lhcb 1 * 3 gene ( 56 ) in the presence of 0 . 5 μg of poly ( di - dc ) at 25 ° c . for 15 min . wce were incubated in preincubation buffer ( 50 mm tris - hcl , ph 7 . 5 , 2 mm mncl 2 , 5 mm dtt , 0 . 1 mm edta , 0 . 01 % brij 35 ) at 30 ° c . for 45 min , then incubated with the a2 probe in the presence of 1 μg of poly ( di - dc ). the emsa buffer and electrophoresis conditions have been described ( 56 ). dna - protein complexes were detected by autoradiography . to isolate proteins that interact with cca1 , the yeast two - hybrid system that uses gal4 recognition sites to regulate expression of both his3 and lacz genes was used ( 21 ). the gal4 - db - cca1 fusion protein did not itself activate transcription of the reporter genes . four positive colonies were obtained which contained plasmid that activated his3 and lacz transcription only in the presence of gal4 - db - cca1 . these fell into two classes based on sequence analysis , and one of them , clone 106 , was fully characterized . the reading frame of clone 106 encoded a 214 amino acid fragment . because a putative translation initiation codon was missing in this cdna clone , corresponding clones were isolated from an arabidopsis cdna library . the sequence of the full - length cdna insert is shown in fig1 a . the 276 amino acid residues open reading frame encodes an estimated 30 . 8 kda protein . the first atg codon of the open reading frame starts at 142 bp and is preceded by an in - frame stop codon at the − 42 to − 40 position and by a purine ( a ) at the − 3 position . this is a favorable context for an initiation codon in plants ( 32 ). the deduced amino acid sequence is highly homologous to the β - subunit of protein kinase ck2 , in particular to arabidopsis ckb1 and ckb2 . thus , the gene corresponding to this cdna clone was designated ckb3 . the nucleic acid sequence is separately illustrated as seq . i . d . no . 1 in fig2 while the amino acid sequence is separately illustrated as seq . i . d . no . 2 in fig3 . fig1 b shows an alignment of the amino acid sequences of arabidopsis ckb1 . ckb2 , and ckb3 . the amino acid identities between ckb3 and ckb1 , ckb3 and ckb2 , and ckb1 and ckb2 are 75 %, 71 % and 80 %, respectively . the similarity is greatest over the carboxyl - terminal two thirds of the three proteins . the ckb3 protein shares most of the structural features of ckb1 and ckb2 at the level of primary structure ( 10 ). first , ckb3 contains a potential metal - binding motif cys - pro - x 3 - cys - x 22 - cys - pro - x - cys ( seq . i . d . no . 3 ) ( 45 ) ( wherein “ x ” may be any amino acid ). second , although the conserved autophosphorylation site , ser - ser - ser - glu - glu ( seq . i . d . no . 4 ), is missing in the amino - terminal region of ckb3 , there are two ck2 recognition phosphorylation sites . 81 ser - gly - ser - glu - gly - asp ( seq i . d . no . 5 ) and 83 ser - glu - gly - asp - asp , ( seq i . d . no . 6 ) in about the same location as in the animal β - subunits . third , ckb3 has an n - terminal extension preceding the putative phosphorylation sites which exhibits a moderate level of similarity to the n - terminal extension of the other arabidopsis β - subunits . neither yeast nor animal β - subunits contain such an n - terminal extension , and this region bears no extensive similarity to other proteins . s . cerevisiae has two genes coding for the catalytic ( α ) subunits of ck2 , and at least one of the two genes is required for vegetative growth . ydh8 , which carries the cka1 - δ1 cka2 - 8 mutation , grows at 25 ° c ., but not at 35 . 5 ° c . ( 20 ), and this temperature sensitivity can be compensated for by overexpressing ck2 β - subunits , including arabidopsis ckb1 and ckb2 ( 10 ). we tested whether ckb3 could also compensate for the temperature sensitivity of the mutation . as controls , pkt10 and pjcr14 , which contains the s . cerevisiae ckb2 gene , were also transformed into ydh8 . fig4 shows that ydh8 cells expressing either s . cerevisiae ckb2 or arabidopsis ckb3 could grow both at 25 ° c . and 35 . 5 ° c ., while transformants with pkt10 could grow only at 25 ° c . these results demonstrate that ckb3 shares functional similarity with ckb1 and ckb2 . although ckb1 , ckb2 , and ckb3 share a high degree of structural and functional homology , it might be that ckb3 is the only β - subunit that associates with cca1 specifically . therefore , we tested whether ckb1 and / or ckb2 are also able to interact with cca1 in the yeast two - hybrid system and confirmed that the full - length ckb3 could interact with cca1 in the same way as the product of the original cdna clone . fig5 a demonstrates that ckb1 , ckb2 and ckb3 can each interact specifically with cca1 in yeast . to further investigate the direct interaction of cca1 with β - subunits of ck2 , the ability of gst - cca1 to bind to ck2 β - subunits in vitro was also tested . fig5 b shows that ckb1 and ckb2 bound to gst - cca1 efficiently but not to gst alone ( lanes 4 and 7 ). ckb3 also interacted with gst - cca1 , but apparently less efficiently than did ckb1 and ckb2 ( lane 8 ). similar analyses showed that cka1 and cka2 , the two α - subunits of ck2 ( 39 ), also bound to gst - cca1 ( lane 9 ). while it is possible that ck2 β - subunits that could be present in the wheat germ extract might mediate the interaction of cca1 with the ck2 α - subunit , our results suggest that cca1 can interact with both ck2 α - subunits and β - subunits in vitro . as a negative control in these experiments , gbf4 , a bzip transcription factor ( 35 ), was used . gbf4 did not show a specific interaction with gst - cca1 ( lanes 11 and 12 ), confirming that the interaction of cca1 with ck2 subunits is specific . ck2 can stimulate binding of cca1 to the lhcb1 * 3 promoter in vitro . the possible biochemical consequences of the interaction of ck2 β - subunits with cca1 were examined . first , we tested whether dna binding activity of cca1 was affected by its interaction with ck2 β - subunits . as shown in fig6 binding of cca1 to the a2 fragment was stimulated specifically by ckb3 at low concentrations of cca1 ( lanes 3 - 7 ). ckb3 did not itself show any binding to the probe ( lane 2 ) ckb1 also enhanced dna binding activity of cca1 in the same way ( lanes 10 - 14 ). a second approach to understand the function of the ck2 - cca1 interaction was to determine whether recombinant ck2 can phosphorylate cca1 in vitro . cca1 has several putative sites for phosphorylation by ck2 . we initially tested phosphorylation of cca1 by cka1 , one of the α - subunits of ck2 . fig7 a shows that a large amount of cka1 ( 280 ng ) phosphorylates gst - cca1 but not gst ( lanes 1 and 3 ). it has been shown that ck2 β - subunits stimulate the catalytic activity of α - subunits toward most substrates ( 42 , 24 , 1 ,). fig7 b shows that when a smaller amount ( 14 ng ) of cka1 was used , a strong stimulation of the cca1 phosphorylation was observed by adding either ckb1 or ckb3 ( lanes 2 - 4 ). similar results were obtained for cka2 , the other α - subunit of ck2 ( data not shown ). fig7 b also shows that cca1 could be phosphorylated in the presence of gtp as well as in the presence of atp ( lanes 5 - 7 ). these data confirm that the phosphorylation can be attributed to ck2 activity , because ck2 is unique among protein kinases in that it can use both atp and gtp as a phosphodonor . phosphorylation by ck2 has been shown to affect the dna binding activity of many transcription factors . therefore , the possible effect of ck2 phosphorylation on the dna binding activity of cca1 was examined . when recombinant cca1 was phosphorylated by ck2 , no effect on its dna binding activity was observed in the emsa assay ( data not shown ). arabidopsis plants contain a ck2 - like activity that can phosphorylate cca1 in vitro . we next examined whether plants contain a ck2 - like protein kinase activity that can phosphorylate cca1 in vitro . fig8 shows that gst - cca1 , but not gst alone , was phosphorylated by a kinase activity in arabidopsis whole - cell extracts in vitro ( lanes 1 and 2 ). fig8 also shows that this kinase activity was able to utilize both atp and gtp as a phosphodonor ( lane 5 ). furthermore , addition of 2 , 3 - diphosphoglycerate , which is an inhibitor of ck2 ( 18 ), reduced the incorporation of atp into gst - cca1 by 63 % ( lane 3 ). when this inhibitor was added to recombinant ck2 , the phosphorylation of cca1 was reduced by 77 % ( lane 7 ). these results demonstrate that the arabidopsis plants contain a ck2 - like activity that phosphorylates cca1 in vitro , and that this kinase activity is responsible for much of the phosphorylating activity on cca1 in the extracts . phosphorylation by ck2 is required for formation of the dna - protein complex containing cca1 in plant extracts . fig9 a shows that the dna binding activity of the major cca1 - containing complex in plant extracts depends on phosphorylation . when the extracts were treated with λ protein phosphatase , formation of the complex was inhibited completely . the major dna - protein complex containing cca1 ( marked with arrows ) migrated more slowly than that formed with recombinant cca1 , suggesting that other proteins in the plant extracts are included in this complex , interacting with cca1 and / or binding to the dna . the a2 fragment used as a probe includes known binding sites for g - box and caat binding proteins ( 56 ). fig9 b shows that this complex is more abundant in extracts of plants expressing cca1 under the control of a constitutive promoter ( 62 ). further evidence that the marked complex contains cca1 includes the observations ( data not shown ) that this complex is absent in extracts prepared from cca1 - null mutant plants and that addition of anti - cca1 antibodies inhibits its formation . fig9 c demonstrates that the ck2 - like phosphorylation activity in the extracts is important for this binding activity . formation of the complex was abolished when ck2 inhibitors , either 2 , 3 - diphosphoglycerate or quercetin , were added to the preincubation reactions . 2 , 3 - diphosphoglycerate inhibits both ck1 and ck2 , whereas inhibition by quercetin is specific to ck2 ( 18 , 9 ). the components of the new bands that appear in the inhibitor treated samples are not yet known , but these bands presumably represent a change in the composition of the cca1 - containing complex in the absence of gk2 - mediated phosphorylation . taken together , these results demonstrate that phosphorylation by ck2 is required in the plant extracts for formation of the major dna - protein complex containing cca1 . the protein kinase ck2 is a ser / thr kinase that is ubiquitously expressed and highly conserved ( 42 , 24 , 1 ). ck2 consists of two catalytic ( α ) and two regulatory ( β ) subunits , which form an α 2 β 2 heteromeric holoenzyme . although most organisms have two genes encoding α - subunits and one gene encoding the β - subunit , two genes encoding β - subunits have been reported in s . cerevisiae and arabidopsis ( 45 , 10 , 3 ). the ckb3 protein exhibits significant amino acid sequence identity with arabidopsis ckb1 and ckb2 . several lines of evidence confirm that ckb3 does indeed function as a third ck2 β - subunit in arabidopsis . first , ckb3 was able to compensate for the temperature - sensitive growth defect of an s . cerevisiae cka1 - δ1 cka2 - 8 mutation . second , recombinant ckb3 was able to stimulate the catalytic activity of cka1 when cca1 was used as a substrate . ckb3 is the only reported example of a third ck2 β - subunit in any organism . interestingly , it had been suggested previously that there might be a third ck2 α - subunit in arabidopsis ( 39 ). it is yet to be determined whether there are several forms of the holoenzyme with different subunit compositions or whether different subunits confer different substrate specificities and / or tissue specificities . we have shown that ck2 β - subunits specifically interact with cca1 both in yeast and in an in vitro interaction assay . we did not isolate clones for the two other ck2 β - subunits in the initial screen , but we have observed that the growth of yeast containing the constructs for these subunits is slower than that of cells expressing the cal4 - ad - ckb3 construct , and this may account for our failure to identify them along with ckb3 . we have found that recombinant his - tagged ck2 β - subunits stimulate binding of cca1 to a fragment of the lhcb1 * 3 gene . this effect is likely to be specific because obp1 , a dna binding protein that stimulates interaction of obf4 and obf5 with ocs elements , did not affect binding of cca1 to the a2 fragment ( 66 ). also , both recombinant gst - ckb1 and gst - ckb3 stimulated dna binding of cca1 , whereas gst alone had no effect ( data not shown ), demonstrating that ck2 β - subunits are responsible for the enhancement . the fact that the mobility of the complex was not affected suggests that the interaction of the proteins might be transient or unstable under the conditions for the emsa . there have been other such reports of enhancement of dna binding by a second protein without altering the mobility of the dna - protein complex ( 66 , 60 , 31 , 11 ). the fact that ck2 β - subunits associate with cca1 and stimulate its binding to the lhcb1 * 3 promoter suggests a different mechanism for regulation of cca1 dna binding activity other than phosphorylation . in fact , the α - subunit is not required for this stimulation , and ck2 β - subunits cannot themselves phosphorylate cca1 . it is possible that in addition to being the regulatory subunit of ck2 , the β - subunit might play other roles in the cell . overexpression of the ck2 β - subunit in schizosaccharomyces pombe causes multiple septation and inhibits cell growth and cytokinesis ( 47 ). these phenotypes appeared to be due to the production of free β - subunit rather than to excess holoenzyme . in xenopus oocytes , the β - subunit interacts with mos , a germ cell - specific ser / thr kinase that is required for oocyte maturation , and this interaction negatively regulates mos - mediated mitogen - activated protein kinase activation resulting in repression of oocyte maturation ( 7 , 6 ). recently , it was also shown that cyclin d , which is a regulatory component of complexes of cyclin with cyclin dependent kinase ( cdk ), stimulates transcriptional activity of estrogen receptor independent of interaction with cdks ( 67 ). therefore , it is intriguing to speculate that direct interaction of ck2 β - subunits with cca1 stimulates binding of cca1 to promoter sequences and can affect cca1 - mediated transcription . we have demonstrated that cka1 phosphorylates cca1 in vitro and that both ckb1 and ckb3 stimulate this phosphorylation . although cka1ckb1 showed a higher activity of cca1 phosphorylation than cka1ckb3 , the possibility that this was due to differing relative activities of ckb1 and ckb3 in their corresponding preparations cannot be excluded . we have also demonstrated that arabidopsis plants contain a ck2 - like protein kinase activity that can phosphorylate cca1 in vitro , and that this is a major kinase activity for cca1 phosphorylation in the extracts . the identity of this kinase was confirmed in two ways . first , the kinase activity phosphorylates cca1 in the presence of gtp as well as atp , a unique characteristic that distinguishes ck2 from other ser / thr kinases . second , addition of 2 , 3 ,- diphosphoglycerate , an inhibitor of ck2 , inhibited most of the cca1 phosphorylating activity in the plant extracts . phosphorylation of transcription factors by ck2 has been reported to modulate their dna binding activity , cellular localization , metabolism , and interaction with other proteins ( 1 , 12 , 28 , 40 , 2 , 25 , 34 , 41 ). it was recently shown that the lhcb1 * 1 rna level in transgenic plants overexpressing cca1 decreased steadily when plants were transferred from light - dark cycles into constant dark even though cca1 was expressed at a high level ( 62 ). we have also observed that the lhcb1 * 1 rna level in etiolated transgenic plants overexpressing cca1 was as low as that in etiolated wild type plants ( data not shown ). these observations suggest that cca1 activity is regulated by light through posttranslational modifications , one of which could be phosphorylation . in this regard , our finding that plant extracts contain a ck2 - like activity that is required for formation of the major dna - protein complex containing cca1 is especially noteworthy . the cca1 - containing complex is likely to contain a protein or proteins in addition to cca1 . although ck2 phosphorylation of recombinant cca1 did not affect its dna binding acitvity in vitro , it is possible that in the plants the other proteins affect the relative binding affinities of the phosphorylated and non - phosphorylated forms of cca1 for its binding sites . alternatively , the phosphorylation state of cca1 might be important for protein - protein interactions of cca1 with other protein ( s ) in the complex , it is also possible that phosphorylation of other protein ( s ) by ck2 is essential for the cca1 complex formation . to further explore the hypothesis that the ck2 - cca1 dna - protein complex plays a role in the regulation of the circadian clock , we created transgenic arabidopsis plants overexpressing a c - myc tagged form of ckb3 and analyzed their circadian behavior . to produce the tagged ckb3 an eco r [- bsr g ] fragment of the plasmid puc - ckb3 that contains the entire coding sequence of ckb3 cdna at the bam hi site of puc19 was replaced with the duplex dna composed of oligonucleotide myc - ckb3f ( 5 ′- aattgagatctcatggagcaaaagcttatcagcgaggaggactt gaacat ) ( seq i . d . no . 7 ) and oligo - nucleotide myc - ckb3b ( 5 ′- gtacatgttcaagtcctcctc gctgataagcttttgctccatgagatct ) ( seq i . d . no . 8 ) to introduce the bgl ii site and c - myc encoding sequence in front of ckb3 . the resultant plasmid was digested with bgl ii and hinc ii , and the bgl ii - hinc ii fragment was subcloned into the pbii21 vector ( clonetech ). this construct was used to transform agrobacterium tumefaciens strain a2260 , and then arabidopsis plants ( columbia ecotype ) using the in planta transformation procedure as described ( 61 , 62 ). overexpression of ckb3 had no apparent effects on plant growth and development except timing of flowering . from 16 transgenic lines that each had a single site of insertion , two transgenic lines designated ox18 and ox41 were further analyzed . levels of ckb3 transcript in the fourth generation of homozygous ckb3 - overexpressing ( ckb3 - ox ) plants were approximately 20 times higher than that in the wild - type ( fig1 a ). ten μg of total rna were treated with rq1 rnase - free dnase ( promega ) and the first - strand cdna was synthesized as described in ( 43 ) the product of the first - strand synthesis was then used for pcr to amplify 140 bp ckb3 cdna with the primers ckb3f1 ( 5 ′- acaaggaacgtagtggaggaggtg ) ( seq i . d . no . 9 ) and ckb3b3 ( 5 ′- aaccctagatgt ggtggtggaag ) ( seq i . d . no . 10 ). as a control , primers ubq10 - 5 ′ and ubq10 - 3 ′ ( 61 , 62 ) were used to amplify 111 bp ubq10 cdna . the resultant pcr fragments were separated on a 2 % agarose gel , blotted and hybridized with 32 p - labeled probes . the transgenic plants contained appreciable amounts of the c - myc - tagged ckb3 protein ( fig1 b ). protein extracts were obtained by grinding 10 - day - old seedlings in 100 μl of 3xsds - sample buffer ( 180 mm tris - hcl ( ph 6 . 8 ), 6 % sds , 30 % glycerol , 7 . 5 % 2 - mercaptoethanol ), boiling this mixture for 5 min and saving the supernatant after centrifugation for 15 min at 14 , 000 × g . protein concentration was measured with a protein assay reagent ( bio - rad ). western blots were performed using anti - c - myc monoclonal antibody 9e10 ( 17 , 15 , 64 , 46 ) following the methods described in ( 61 , 62 ). measurement of ck2 activity in whole - cell extracts showed that the transgenic lines exhibited a 1 . 7 - fold increase in ck2 activity ( fig1 c ). frozen seedlings ( 100 mg ) were ground and extracted with 100 μl of extraction buffer ( 50 mm tris - hcl ( ph 7 . 5 ). 15 mm mgcl 2 , 0 . 1 m kcl , 0 . 25 m sucrose and 10 % glycerol , 1 mm phenylmethylsulfonylfluoride , protease inhibitor cocktail ( boehringer mannheim ), phosphatase inhibitor mixture ( 55 , 26 , 48 ) and 14 mm 2 - mercaptoethanol ). after centrifugation at 14 , 000 × g for 15 min . the supernatant was saved and protein concentration was measured as above . ck2 assays were carried out at 37 ° c . with 200 μm ck2 specific peptide substrate arg - arg - arg - asp - asp - asp - ser - asp - asp - asp ( seq i . d . no . 11 ) ( boehringer mannheim ) in 25 μl of ck2 buffer ( 55 , 26 , 48 ) as described ( 5 ). we next examined whether ckb3 overexpression affected circadian expression of the cca1 and lhy genes . oscillations in expression of these genes are robust in wild - type plants transferred into continuous light . fig1 a and 11b show that the periods of cca1 and lhy rnas were shortened by about four hours in the ckb3 - ox plants ( total rna isolation and rna blot analyses were performed as described in ( 61 , 62 , 19 )), and no differences in the amplitude of the rhythms were seen . fig1 c shows that the period of the cca1 protein oscillation was also shortened ( immunoblot analyses of the cca1 protein were carried out as explained in ( 61 , 62 )). thus , overexpression of ckb3 affected circadian rhythms of these two genes that are closely associated with the circadian clock . because lhy is closely related to cca1 both structurally and functionally ( 49 ), we tested the possibility that ck2 can also interact with and phosphorylate lhy . fig1 a shows that , like cca1 , lhy could bind to ckb1 and ckb3 efficiently and specifically . to make this determination a pcr fragment containing the entire lhy gene was cloned in pgex - 3x ( pharmacia ). the gst - lhy fusion protein was produced and purified as described in ( 55 , 26 , 48 ). in vitro binding assays were performed using 35 s - labeled ckb3 and glutathione - agarose beads ( sigma ) with bound gst or gst - lhy . furthermore , fig1 b shows that cka1 , the α - subunit of ck2 , could phosphorylate lhy , and this phosphorylation was enhanced by adding either ckb1 or ckb3 . in vitro kinase assays were performed using gst - lhy bound to glutathione - agarose beads and recombinant ck2α - and β - subunits as described in ( 55 , 26 , 48 ). these data are consistent with the idea that ck2 can interact with and phosphorylate both cca1 and lhy in arabidopsis . if the function of both cca1 and lhy is closely associated with a central oscillator and this is altered by increased ck2 activity , then the period lengths of the circadian rhythms of output genes should also be changed to reflect that of the cca1 and lhy rna rhythms . we therefore tested whether overexpression of ckb3 affected genes representing different rhythmic outputs of the circadian clock . lhcb1 * 1 and cat2 rnas normally peak around subjective dawn and during the subjective day , whereas cat3 and ccr2 rnas peak considerably later in wild - type plants . fig1 shows that overexpression of ckb3 had the same effect on the circadian expression of these genes as it did on the cca1 and lhy rna rhythms . although it did not alter the amplitudes , it shortened the periods of the rhythms by about four hours . it has been shown that the circadian clock is involved in the control of hypocotyl elongation and the photoperiodic flowering response ( 13 ). overexpression of ckb3 did not affect hypocotyl elongation under continuous white light or dark conditions . hypocotyl lengths of 5 - day - old seedlings were measured using a digital camera ( kodak dcs 420 ) and the nih image program . the average hypocotyl lengths of plants grown in continuous white light were : wild - type , 2 . 19 ± 0 . 09 mm ; ckb3 - ox , 2 . 23 ± 0 . 14 mm ( line ox18 ) and 2 . 06 ± 0 . 85 mm ( line ox41 ); in the dark : wild - type , 11 . 6 ± 0 . 28 ; ckb3 - ox , 11 . 3 ± 0 . 30 mm ( line ox18 ) and 11 . 6 ± 0 . 28 mm ( line ox41 ). however , ckb3 overexpression did affect the photoperiodic induction of flowering . table 1 shows that ckb3 - ox lines flowered earlier than wild - type in short - day conditions , whereas ckb3 overexpression did not substantially affect flowering time in long - day conditions . to obtain data for the table plants were grown under long - day ( l : d 16 : 8 ) or short - day ( l : d 8 : 16 ) conditions as described in ( 20 ). the table shows the number of total leaves ( including cauline leaves on the main stem ) on the day when the first flower opened . leaves of 10 - 20 plants were counted and values reported are means ± standard error . experiments were done three times under long - day and twice under short - day conditions with similar results . under long - day conditions both the experimental and wild type plants have nearly the same number of leaves showing that flowering response was essentially unaltered . however , under short - day conditions the wild type plants take much longer to flower growing significantly larger ( more leaves ) than the experimental plants . thus , ckb3 overexpression caused a diminished photoperiodic flowering response , and while we cannot rule out other possibilities , this effect may well be a result of the altered clock function in ckb3 - ox plants . these data show that an increase in ck2 activity alters circadian rhythms in arabidopsis . ck2 can interact with and phosphorylate both cca1 and lhy , proteins that are closely associated with the central oscillator of the circadian clock . overexpression of ckb3 substantially increased the catalytic activity of ck2 in the plant in a way similar to that seen in other organisms ( 47 ). this overexpression in arabidopsis caused shorter periods of circadian oscillations of both cca1 and lhy rnas as well as those of several output genes . it also affected the timing of flowering , but did not affect hypocotyl elongation . in these respects , the ckb3 - ox lines are similar to phenotypes of toc1 , as both lines exhibited shorter periods of output genes , a reduced photoperiodic flowering response in the columbia ecotype , but normal hypocotyl elongation under light and dark conditions ( 37 , 52 ). two classes of mutants which affect the input pathway , det1 and plants overexpressing phya and phyb , also exhibited a short - period phenotype ( 4 , 8 , 38 , 53 ). however , morphological phenotypes that are also associated with these lines are absent in the ckb3 - ox and toc1 plants . furthermore , in contrast to the higher lhcb1 * 1 expression seen in the det1 and phya / b - overexpressing lines , induction of lhcb1 * 1 in ckb3 - ox plants by brief red illumination was reduced compared to wild - type ( data not shown ). this observation is consistent with the finding that antisense expression of the ck2 α - subunit gene increased the expression of lhcb1 * 1 under similar conditions ( 30 ). we conclude that ck2 affects components that are part of the central oscillator itself or closely associated with it . recently , mutant alleles of a clock gene , double - time ( dbt ), were isolated in drosophila ( 29 ). these mutations alter the periods of behavioral rhythms and molecular oscillations of clock components per and tim and also affect per phosphorylation and stability . the dbt gene encodes a protein ( dbt ) closely related to human casein kinase 1ε ( 29 ). furthermore , dbt can interact with per in vitro and in drosophila cells , suggesting that dbt regulates per phosphorylation directly . both casein kinase 1ε and ck2 are ser / thr kinases that do not require a second messenger as a cofactor , and both prefer acidic substrates such as casein ( 42 , 54 , 24 ). our finding that ck2 can interact with and phosphorylate both cca1 and lhy in vitro is consistent with the idea that ck2 could modulate the circadian clock by direct interaction with and / or phosphorylation of the cca1 and lhy proteins . such interactions may control the activity and / or stability of these proteins . we have found that cca1 and lhy can interact in vitro ( data not shown ) and such an interaction could also be a target for modification by ck2 . because the periods of lhy and several output genes expression were also shortened in a cca1 - null mutant line ( 19 ), we suggest that ckb3 overexpression might inactivate and / or destabilize cca1 and lhy by altering their phosphorylation state . we cannot exclude the possibility that ckb3 affects clock components other than or in addition to cca1 and lhy . however , the results presented here clearly demonstrate that the protein kinase ck2 is involved in the function of the circadian clock in arabidopsis . in summary , interaction of subunits of ck2 with cca1 and phosphorylation of cca1 by ck2 may modulate cca1 activities that are required for phytochrome regulation of lhcb1 * 3 gene expression and for circadian clock function . in light of the involvement of cca1 in circadian rhythms ( 62 ), it is of particular interest that the clock gene affected in the double - time mutant of drosophila and which is required for circadian rhythmicity has recently been cloned and found to be closely related to human casein kinase 1ε ( 29 ). while the physiological significance of the ck2 - 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