because they are sessile , plants have evolved to respond adaptively 
 to their ever - changing light environment by photoreceptor - mediated 
 physiological responses .
plants possess several families of photoreceptors 
 that can sense light direction , duration , quality , and quantity , including 
 red and far - red photoreceptors ( phytochromes ) and three classes of 
 uv - a / blue photoreceptors ( cryptochromes , phototropins , and members 
 of the zeitlupe family ) .
these photoreceptors 
 use light signals to regulate almost every phase of plant growth and 
 development , from seed germination through flowering and senescence . 

for example , phototropins mediate a number of blue light responses 
 in arabidopsis thaliana , including 
 phototropism , chloroplast movement , leaf flattening , leaf positioning , 
 stomatal opening , and rapid inhibition of growth of dark - grown seedlings , thus optimizing light capture for plant photosynthesis 
 and growth .
fifteen years ago , christie et al . first 
 demonstrated that phototropin 1 ( phot1 ) acts as a blue light / uv - a 
 receptor for phototropism in land plants .
this membrane - associated 
 photoreceptor was the first of a family of two ( phot1 and phot2 ) , 
 and a great deal has been learned about its structure and photochemistry .
its two flavin - binding domains ( designated lov1 and lov2 ) have been 
 extensively studied by flash photolysis , ftir , resonance raman spectroscopy , 
 and absorption and fluorescence spectroscopy , among other biophysical 
 techniques . with the discovery that lov 
 domain - containing proteins are widespread in all major groups of bacteria 
 ( including archaea ) ,
all three major groups of fungi , and all orders 
 of green plants , the lov1 and lov2 domains 
 from phot1 of avena sativa ( domestic 
 oats ) have served as prototypes for investigating lov domains from 
 a wide range of organisms .
the structures of several lov domains have been obtained by x - ray 
 crystallography , and a solution structure 
 of oat phot1 lov2 has been obtained by nmr .
these combined studies indicate that lov domains consist of five 
 antiparallel  sheets separated by short  helices .
downstream 
 of lov2 is an amphipathic  helix ( designated the j- 
 helix ) .
on photoexcitation of the flavin chromophore , 
 the j- helix is released from the  sheets and loses 
 its coiled structure , and the structural change presumably activates 
 the downstream kinase moiety in phototropins .
substitution of hydrophilic 
 amino acids on the hydrophobic side of the j- helix leads to 
 constitutive activation of the kinase function .
indeed , the single 
 lov domain in aureochrome from the stramenopile algae actually activates 
 a bzip domain that is upstream of the lov domain , not downstream .
hence , photoexcitation of lov domain proteins 
 can lead to more than one type of conformational change in the protein .
although much is known about the biochemical and photophysiological 
 properties of the phototropins , progress has been considerably slower 
 in elucidating the post - translational modifications of these photoreceptors . 

it has been known since the earliest studies that light activates 
 the phosphorylation of multiple sites on a protein that was subsequently identified as phot1 .
salomon et al . showed that 
 the phosphorylation was in some way hierarchical , with certain sites 
 phosphorylated at low blue light fluences and other sites phosphorylated 
 only at higher blue light fluences .
the same study demonstrated the 
 reverse pattern during a period in darkness as the phototropin returned 
 to its dark state after irradiation .
more recently , two different 
 studies identified a number of specific sites that become phosphorylated 
 upon light activation .
finally , roberts et al . demonstrated that phot1 from arabidopsis is monoubiquitinated in vivo in response 
 to low fluences of blue light and multi- and/or polyubiquitinated 
 in response to high fluences of blue light .
any further characterization of the full - length phototropins has 
 been severely hindered by the lack of success in producing sufficient 
 amounts of highly purified photoreceptors for structural , biochemical , 
 or biophysical studies .
here , we use two - dimensional difference gel 
 electrophoresis ( 2d dige ) to examine any dynamic changes in mass or 
 charge occurring in full - length arabidopsis phot1 in vivo during photoexcitation and subsequent dark recovery . 

we characterize phot1 in its stable dark state as well as its state 
 immediately after saturating light treatment and the completion of 
 phosphorylation .
in addition , we employed a combination of immunoprecipitation 
 and mass spectrometry analysis to search for any additional post - translational 
 modifications that have not been previously identified .
in this study , a. thaliana columbia ( col-0 ) seedlings and transgenic arabidopsis expressing phot1gfp in a phot15 background were 
 used . for etiolated seedlings , seeds were surface - sterilized and sown 
 on ms plates ( half - strength ms medium , 0.8% agar , 43.8 mm sucrose , ph 5.7 ) , cold - treated ( 2 days at 4 c ) 
 in the dark , exposed to white light of medium intensity ( 100 mol 
 photons m s ) for 6 h , and 
 then incubated in the dark growth room for 4 days at 22 c .
blue 
 light irradiation was performed in a growth chamber ( e-30 led , percival 
 scientific , perry , ia , usa ) with far - red , red , and blue ( 468 nm ) light - emitting 
 diode sources .
the fluence rate was measured using a li-250a light 
 meter with a li-190sa quantum sensor ( li - cor , lincon , ne , usa ) .
etiolated 
 seedlings were irradiated for up to 60 min with continuous blue light 
 ( 20 mol m ) or left in darkness as controls . 

the whole seedlings , including the cotyledons , hypocotyls , and roots , 
 were collected and frozen immediately in liquid nitrogen until protein 
 extraction and proteomic analyses were performed ( figure 1 ) .
schematic documentation of the experimental design ( a ) and a photograph 
 of a representative etiolated arabidopsis seedling ( b ) .
four - day - old etiolated seedlings of arabidopsis thaliana ( b ) were used and were either 
 left in darkness ( control ) or irradiated with blue light .
thereafter , 
 entire seedlings were used for protein extraction and proteomic analyses , 
 which were combined with mass spectrometric and immunoblot analyses . 
 note that blue light treatment was from the top .
the white bar is 
 1 mm in panel b. microsomal proteins were extracted 
 as described in previous studies .
briefly , liquid - ground 
 tissue powder was mixed with three volumes of extraction buffer ( 50 
 mm hepes , ph 7.5 , 0.33 m sucrose , 5% ( v / v ) glycerol , 3 mm edta , and 
 5 mm dtt ) with phosphatase and protease inhibitors ( 10 mm sodium fluoride , 
 10 mm sodium molybdate , 10 mm imidazole , 1 mm activated sodium vanadate , 
 7 m e-64 , 1.5 m bestatin , 2 m pepstatin , 4 m 
 antipain , and 1 mm pmsf ) and then centrifuged at 10  000 g for 20 min to remove cell debris .
the resulting supernatant 
 was centrifuged at 200  000 g for 60 min to 
 pellet the microsomal fraction .
the microsomal pellet was dissolved 
 in sds extraction buffer ( 100 mm tris - hcl , ph 8.0 , 2% sds , 1% -mercaptoethanol , 
 5 mm egta , and 10 mm edta ) and further purified with a modified phenol 
the protein extract 
 was dissolved in 2d dige buffer ( 6 m urea , 2 m thiourea , and 4% chaps ) 
 and quantified using the bio - rad protein assay .
twenty micrograms of microsomal proteins from 
 dark - control or blue light - treated plants ( ph 8.5 ) were mixed with 
 80 pmol of cy3 or cy5 dyes and incubated on ice for at least 2 h in 
 the dark .
cy3- and cy5-labeled proteins were then combined 
 and used for isoelectric focusing ( ief ) .
ief was performed on 24 cm 
 ipg strips , ph 310 nl ( ge healthcare , piscataway , nj , usa ) . 
 the running conditions were as follows : rehydration for 2 h , 50 v 
 for 10 h , step and hold at 500 v and then 1000 v for 1 h each , gradient 
 to 8000 v over 3 h , and then at 8000 v until reaching a total of 56  000 
 v h. second - dimension electrophoresis was performed using 10% sds - polyacrylamide 
 gels .
the electrophoresis was performed at 40 v for 2 h and then at 
 120 v until the bromophenol blue front reached the bottom of the gel . 

images of cy3- and cy5-labeled proteins were acquired using a typhoon 
 trio scanner ( ge healthcare ) . the estimated ph ranges following ief 
 are indicated in figures 24 .
a differential in - gel analysis module 
 with an estimated spot number of 5000 was used for spot detection , 
 and a biological variation analysis module was used to identify spots 
 differentially regulated by blue light ( p value < 
 0.05 ) .
four biological replicates were used in each comparison , and 
 spots of interests were manually checked to confirm spot matching 
 between different gels and to remove artifacts . spot 
 picking and reverse - phase liquid chromatography 
electrospray 
 tandem mass spectrometry ( lc  ms / ms ) analyses were performed 
 as follows .
approximately 400 g of protein was labeled with 
 40 pmol of cy5 dye and separated by 2-de .
after electrophoresis , 2-de 
 gels were stained with deep purple stain ( ge healthcare ) . upon scanning 
 the gels with typhoon trio ,
spots of interests were selected with 
 decyder software and picked by an ettan spot picker ( ge healthcare ) . 

the excised protein spots were washed twice with 50% acetonitrile 
 in 25 mm ammonium bicarbonate ( nh4hco3 ) , vacuum - dried , 
 rehydrated in 10 l of digestion buffer ( 10 ng/l trypsin 
 in 25 mm nh4hco3 ) , and covered with a minimum 
 volume of nh4hco3 .
after overnight digestion 
 at 37 c , peptides were extracted twice with a solution containing 
 50% ( v / v ) acetonitrile and 5% ( v / v ) formic acid .
the extracted digests 
 were vacuum - dried and resuspended in 10 l of 0.1% formic acid 
 in water and then analyzed by lc 
the digests were separated 
 by nanoflow liquid chromatography using a 100 m  150 
 mm reverse - phase ultra 120 m c18q column ( peeke scientific , 
 redwood city , ca , usa ) at a flow rate of 350 nl / min in an agilent 
 1100 high - performance liquid chromatography system ( agilent technologies , 
 inc .
mobile phase a was 0.1% formic acid in 
 water , and mobile phase b was 0.1% formic acid in acetonitrile .
following 
 equilibration of the column in 2% solvent b , one - half of each digest 
 ( 5 l ) was injected , and then the organic content of the mobile 
 phase was increased linearly to 40% over 30 min and then to 50% in 
 3 min .
the liquid chromatography eluate was coupled to a microionspray 
 source attached to the mass spectrometer .
the following mass spectrometers 
 were used for the analysis of the samples : qstar pulsar ( applied biosystems / mds 
 sciex , south san francisco , ca , usa ) , qstar elite ( applied biosystems ) , 
 ltq - ft icr ( thermo scientific , san jose , ca , usa ) , and ltq orbitrap 
 xl ( thermo scientific ) .
peptides were analyzed in positive ion mode 
 and in information - dependent acquisition mode to switch automatically 
 between ms and ms / ms acquisition . for experiments using the qstar 
 pulsar and qstar elite , ms spectra
were acquired for 1 s in the m / z range between 310 and 1400 .
ms acquisitions 
 were followed by 3 s collision - induced dissociation ( cid ) experiments 
 in information - dependent acquisition mode . for each ms spectrum , the 
 most intense
multiply charged peaks over a threshold of 30 counts 
 were selected for generation of cid mass spectra .
the cid collision 
 energy was automatically set according to the mass - to - charge ( m / z ) ratio and charge state of the precursor 
 ion .
a dynamic exclusion window was applied that prevented the same m / z from being selected for 60 s after 
 its acquisition . for experiments using the ltq orbitrap xl and ltq 
 ft icr ,
ms spectra were acquired in profile mode using the orbitrap 
 or icr analyzers in the m / z range 
 between 310 and 1600 . for each ms spectrum ,
the six most intense multiply 
 charged ions over a threshold of 400 counts were selected to perform 
 cid experiments .
cid collision energy was automatically set to 35% . 
 a dynamic exclusion window of 1 da was applied that prevented the 
 same m / z from being selected for 
 60 s after its acquisition .
peak lists from files acquired by the qstar instruments were generated 
 using mascot distiller version 2.1.0.0 ( matrix science , boston , ma , 
 usa ) .
parameters for ms processing were set as follows : peak half - width , 
 0.02 ; data points per dalton , 100 .
parameters for ms / ms data were 
 set as follows : peak half - width , 0.02 ; data points per dalton , 100 . 
 in the case of data acquired in the ltq orbitrap xl and ltq ft thermo 
 scientific instruments , peak lists were generated using pava in - house 
 software , which is based on the rawextract 
 script from xcalibur v2.4 ( thermo fisher scientific ) . in all cases , 
 the peak lists were searched in - house using proteinprospector version 
 5.4.2 ( the public version is available 
 at http://prospector.ucsf.edu ) .
the enzyme specificity was set to trypsin , and the maximum number 
 of missed enzyme cleavages per peptide was set at one .
carbamidomethylation 
 of cysteine was included as a fixed modification ; n - acetylation of 
 the n terminus of the protein , oxidation of methionine , formation 
 of pyro - glu from n - terminal glutamine , phosphorylation of serine , 
 threonine , or tyrosine , and ubiquitination of lysine were all allowed 
 as variable modifications . in searches of ltq - orbitrap xl or ltq - ft 
 data
, the mass tolerance was 30 ppm for precursor and 0.8 da for fragment 
 ions . for qstar data ,
a precursor mass tolerance of 100 ppm and a 
 fragment mass error tolerance of 0.2 da were allowed for the data 
 search .
the peak lists were searched against a subset of the uniprotkb 
 database as of december 15 , 2009 , containing all entries for arabidopsis ( 53  624 entries searched ) .
the 
 false positive rate was estimated by searching the data using a concatenated 
 database that contains the original arabidopsis uniprotkb database as well as a version of each original entry where 
 the sequence was randomized .
in all protein identifications , a minimal 
 protein score of 22 , a peptide score of 15 , and a minimal discriminate 
 score threshold of 0.0 were used for initial identification criteria . 
 the maximum expectation value threshold ( number of different peptides 
 with scores equivalent to or better than the result reported that 
 are expected to occur in the database search by chance ) was set to 
 0.05 for accepting individual spectra and 0.01 for accepting individual 
 proteins .
when several accession numbers in the database corresponding 
 to overlapping sequences of the same polypeptide were identified , 
 the common gene locus and protein name were reported . only proteins 
 with at least two peptides identified
were further considered and 
 reported . to assign the modification site for peptides containing 
 post - translational modifications , the ms / ms spectrum was reinterpreted 
 manually by matching the observed fragment ions to a theoretical fragmentation 
 obtained using ms product ( proteinprospector ) .
phosphatase treatment was performed 
 as described elsewhere . in brief , 50 l 
 of microsomal protein ( approximately 250 g ) in dige buffer 
 was mixed with 5 l of 10% sds .
then , 345 l of deionized 
 water , 50 l of 20 mm mncl2 , and 50 l of 10 
 -protein phosphatase buffer were added sequentially , mixed , 
 and incubated with 200 units of -protein phosphatase enzyme 
 ( new england biolabs , ipswich , ma , usa ) overnight at 30 c
. then , 
 proteins were pelleted by adding five volumes of 0.1 m ammonium acetate 
 in methanol and dissolved in dige buffer .
the microsomal proteins were prepared as described above and were 
 resuspended in the extraction buffer containing 0.1% ( w / v ) triton 
 x-100 .
after centrifugation at 20  000 g for 
 10 min , solubilized proteins were incubated with an anti - gfp antibody 
 bound to protein a sepharose beads ( ge healthcare ) for 1 h. the beads 
 were washed four times with the extraction buffer containing 0.1% 
 triton x-100 and eluted with 2% sds .
microsomal proteins and 
 the immunoprecipitated proteins were resolved on 7.5% sds - page gel , 
 transferred to a nitrocellulose membrane , and then stained with deep 
 purple stain ( ge healthcare ) , as described .
ubiquitination of phot1 was detected by western blot using an anti - ubiquitin 
 antibody ( fk2 , mouse monoclonal , enzo life science , farmingdale , ny , 
 usa ) , which detects both mono- and polyubiquitinylated proteins .
blue 
 light acts as an environmental cue to regulate plant growth and development , 
 but its perception and transduction by plants remains poorly characterized . 
 to understand better the early events of blue light signaling , the 
 experimental protocol illustrated in figure 1
was employed to study the effect of blue light on the proteome of arabidopsis seedlings . here , we used 2d dige coupled 
 with tandem mass spectrometry to identify early blue light - responsive 
 proteins and protein modifications .
four - day - old etiolated seedlings 
 were irradiated with blue light at a fluence rate of 20 mol 
 m s for 20 min , a total fluence 
 that is known to saturate the phosphorylation of phot1 . because blue light - induced phototropism is initiated by membrane - associated 
 receptors phot1 and phot2 , crude membrane proteins ( microsomal fraction ) 
 from blue light - irradiated and unirradiated seedlings were compared 
 by 2d dige .
two rows of high - molecular - weight proteins ( approximately 
 120 kda ) showed clear mobility shifts in which the more acidic ones 
 showed up only after irradiation and exhibited slightly lower electrophoretic 
 mobility , whereas the more basic ones disappeared after irradiation 
 ( figure 2a ) , which is the pattern that one 
 would expect to see for light - induced protein phosphorylation .
2d dige analysis of the blue light response in the microsomal fraction 
 of 4 day old etiolated arabidopsis seedlings . 

( a , b ) col-0 seedlings were irradiated with blue light for 20 min , 
 and microsomal proteins from both control ( unirradiated , proteins 
 labeled with cy3 ) and irradiated seedlings ( proteins labeled with 
 cy5 ) were analyzed by 2d dige .
( a ) superimposed 2d dige image of the 
 upper - half of a gel .
proteins ( in different modification isoforms ) 
 induced by blue light treatment appear as red spots , and those decreased 
 by the treatment appear green , whereas those remaining constant appear 
 yellow .
spots that were characterized by mass spectrometry are highlighted 
 by arrows , and their identities are listed in table 1 .
( b ) zoomed - in 2d dige overlay image ( from a different gel 
 than that in panel a ) showing the blue light - stimulated accumulation 
 of web1 in the microsomal fraction .
( c ) identities of the row of phot1 
 spots were further confirmed by 2d dige analysis of the microsomal 
 protein of the etiolated seedlings of the phot15 ( labeled with cy3 , green ) and gl ( its genetic background , 
 labeled with cy5 , red ) mutants . the estimated ph ranges following 
 ief are indicated above the gel images in figures 24 . in - gel trypsin digestion of an excised spot followed by liquid 
 chromatography 
tandem mass spectrometry ( ms / ms ) identified 
 these spots as phot1 ( figure 2a , arrow 1 , and table 1 ) , a result 
 that was expected because phot1 has an apparent size of 120 kda .
the identities of the row of phot1 spots from the unilluminated 
 control seedlings were further confirmed by 2d dige comparison of phot15 with its genetic background , gl , as these spots disappeared in the phot15 mutant ( figure 2c ) .
two other blue light - responsive 
 protein spots with higher electrophoretic mobility were also identified 
 as phot1 ( figure 2a , arrows 4 and 5 ) , which 
 are likely the partial degradation products of phot1 .
in addition 
 to phot1 , a blue light - responsive protein was identified as web1 ( weak 
 chloroplast movement under blue light 1 , figure 2 , arrows 2 and 3 ) , an acidic high - molecular - weight protein regulating 
 the movement velocity of chloroplast photorelocation .
web1 was previously shown to be predominantly in the soluble 
 fraction of the proteome , with only a little found in the microsomal 
 fractions .
this is in agreement with 
 its low abundance in the microsomal fraction in our study ( figure 2b ) .
the accumulation of web1 protein in the microsomal 
 fraction after blue light irradiation suggests that light induces 
 web1 association with the cell membrane or some other cellular structure . as shown in figure 2a , proteins spots responsive 
 to blue light were screened by decyder software and were further characterized 
 by ms / ms .
the spot volume ratios of blue light - irradiated to untreated 
 ( positive numbers ) or untreated to irradiated ( negative numbers ) and 
 the p values of the student s t - test of the comparison were calculated from three biological replicates . 
 for the ms / ms identification ,
the number of unique peptides , percentage 
 of sequence coverage , and e value ( best expectance value ) for each 
 spot are listed .
the two rows 
 of phot1 spots extended from the basic to the acidic region in the 
 gel shown in figure 2a . to determine whether 
 all of these spots are the result of differential phosphorylation 
 of phot1 , -protein phosphatase treatment , which hydrolyzes 
 the phosphate groups from serine , threonine , tyrosine , and histidine 
 residues , was performed on the microsomal proteins from both dark 
 and blue light - irradiated seedlings . in both cases ,
phosphatase treatment 
 shifted phot1 to the more basic regions and increased its electrophoretic 
 mobility ( figure 3a , b ) .
however , phot1 was 
 still found as a string of spots in the 2-de gels after the phosphatase 
 treatment , possibly the result of incomplete phosphatase digestion 
 and/or post - translational modifications other than phosphorylation 
 ( i.e. , any modifications affecting charge of the protein ) .
phosphorylation is the major form of phot1 post - translational modification , 
 as indicated by -phosphatase treatment .
phosphatase treatment 
 induced shifts of phot1 spots in microsomal protein from both blue 
 light - irradiated ( a ) and control ( b ) seedlings .
on the left ,  blue 
 and blue/pp  indicates the comparison of phosphatase - treated 
 ( cy5 , red ) verse untreated ( cy3 , green ) microsomal proteins from blue 
 light - irradiated seedlings by 2d dige and  dark and dark/pp  
 indicates the comparison of phosphatase treated ( cy5 , red ) verse untreated 
 ( cy3 , green ) proteins from unirradiated seedlings .
proteins that show 
 up in the phosphatase - treated samples appear red , whereas those in 
 the untreated samples are green .
red arrows point to phot1 spots from 
 samples of irradiated seedlings , green arrows point to those from 
 the unirradiated samples , and white arrows point to those from phosphatase - treated 
 proteins .
salomon et al . found that the phosphorylation 
 of phot1 in avena sativa was , in some 
 way , hierarchical , with certain sites phosphorylated at low blue light 
 fluences and other sites phosphorylated at higher blue light fluences . 

we applied 2d dige to study the time - dependent phot1 phosphorylation 
 and dephosphorylation patterns in arabidopsis .
as shown in figure 4a , a 2 min irradiation at a fluence rate of 20 mol 
 m stimulated phot1 phosphorylation , as indicated 
 by the acidic shift .
a 5 min irradiation shifted phot1 further to 
 the more acidic regions and appeared to saturate phot1 phosphorylation , 
 as longer time treatment ( 20 and 60 min , respectively ) did not cause 
 any further spot shifts in the acidic regions . in contrast , dephosphorylation 
 of phot1 in a sequential order was observed when the saturation - illuminated 
 ( 20 min ) seedlings were kept in the dark .
after a 10 min recovery 
 period in darkness , the charges in phot1 were shifted partially toward 
 a more basic pi , as shown on the 2d dige gel , and after 60 min , there 
 were no further shifts in electrophoretic mobility .
time - dependent phot1 phosphorylation ( a ) and dephosphorylation 
 ( b ) after irradiation of dark - grown seedlings .
( a ) etiolated col-0 
 seedlings were irradiated with blue light for the indicated times , 
 and the microsomal proteins from irradiated ( red ) and unirradiated 
 samples ( green ) were compared by 2d dige . shown
( b ) after saturating irradiation for 
 20 min , the etiolated seedlings were kept in the dark .
the subsequent 
 phot1 dephosphorylation was monitored by 2d dige , comparing the microsomal 
 protein from the col-0 seedling collected at the time points indicated 
 with that of the control samples ( collected immediately after irradiation ) .
two previous studies identified eight different in vivo phot1 phosphorylation 
 sites , but the large number of phot1 spots with 
 distinct pi values ( figures 24 ) that were shifted by protein phosphatase treatment 
 ( figure 3 ) suggested the existence of additional 
 phot1 phosphorylation sites . to identify other possible phot1 phosphorylation 
 sites , an anti - gfp antibody was used to immunoprecipitate phot1gfp 
 from the microsomal proteins of both irradiated and unirradiated arabidopsis seedlings expressing phot1gfp , and the immunoprecipitates were analyzed by 
 tandem mass spectrometry after separation by sds - page . in the phot1gfp 
 proteins from dark - grown seedlings , three phosphorylated ser sites 
 were observed ( supporting information figure 1 ) , with all of them located in the n - terminus ( figure 5 ) . in the illuminated samples ,
13 phosphorylation sites were 
 identified ( figure 5 and supporting information figure 1 ) , which were localized both 
 to the n - terminus and the hinge region between lov1 and lov2 ( figure 5 ) , including two sites ( s58 and s170 ) that were 
 also detected in the dark sample .
a representative annotated tandem 
 mass spectrum of phot1 phosphopeptides is shown in figure 6 ( for the identity of the phosphorylation sites , 
 see table 2 ) . in all
because six of the sites 
 ( s58 , s170 , s185 , s350 , s376 , and s410 ) were reported to be phosphrylated 
 in vivo either in the dark or in illuminated samples , we report here eight additional in vivo phosphorylated sites ( indicated 
 in red in figure 5 )
phosphorylated ser / thr 
 sites identified from blue light - treated tissues are shown above the 
 protein box , whereas those identified from the dark tissues are shown 
 below the protein box .
ser / thr sites in black were identified both 
 in this study and in previous studies , those in blue 
 were not identified in this study but were reported previously , and 
 those in red are new sites identified in this study .
phosphorylated 
 s170 was identified from phot1 protein in both dark and irradiated 
 seedlings in this study , but it was identified only from phot1 protein 
 in dark seedlings in a previous study . in summary , eight new unique sites ( s12 , s92 , s141 , t144
, t360 , 
 s406 , s442 , and s450 ) were identified in this study .
representative cid ms / ms spectrum of phot1 obtained from a precursor 
 ion with a m / z value of 910.7604 , corresponding to a phosphorylated peptide spanning the 
 residues s75k98 of phot1 ( theoretical monoisotopic mass value , 
 910.7576 ; error in the mass observed in the precursor 
 ion , 3.1 ppm ) .
phosphorylated serine is shown in the sequence as s. the observed b and y product ion peaks are labeled accordingly , 
 with the subscripts denoting their position in the identified peptide . 

intense ions corresponding to neutral losses from the labeled sequence 
 ions are indicated by * . in the peptide sequence ,
mass spectra for peptides in this table are provided 
 in figure 6 and in supporting information figure l. s * and t * represent 
 phosphorylation sites .
interestingly , sequence alignment showed that 
 s185 ( in phot1 ) or s121 ( in phot2 ) , located in a highly conserved 
 amino acid region just before lov1 domain of phot1/2 , is the only 
 known conserved site phosphorylated in both proteins in vivo ( supporting information figure 2 ) .
ubiquitination of phot1 has 
 been shown to be involved in phototropic responses , but the ubiquitination site(s ) of phot1 have not yet been 
 characterized .
key features for detection of potential ubiquitin attachment 
 sites on tryptic peptides are the missed cleavage of the modified 
 lysine together with a shift of 114 da ( a diglycine moiety , gg ) in 
 both the mass of the precursor ion and the masses of sequence fragment 
 ions in the ms / ms spectrum that would contain the ubiquitin - modified 
 site of the peptide .
these gg remnants result from 
 the cleavage by trypsin of the original ubiquitin in the c - terminal 
 side or r on its c - terminal ggr sequence .
accordingly , a unique ubiquitination site ( k526 ) , localized in the 
 lov2 region , was observed in the spectrum of the peptide f514k527 
 of phot1gfp from blue light - irradiated seedlings ( figure 7 ) , but it was not observed in the dark controls 
 ( data not shown ) . to document that ubiquitination of phot1
was regulated 
 by blue light irradiation , phot1gfp proteins immunoprecipitated 
 by an anti - gfp antibody were immunoblotted with an anti - ubiquitin 
 fk2 antibody that detects both mono- and polyubiquitinylated proteins . 

immunoblot assays show that phot1 ubiquitination was enhanced after 
 blue light irradiation , whereas it was reduced during recovery in 
 the dark , in a pattern similar to that of phot1 phosphorylation ( figure 8) .
cid ms / ms spectrum obtained from a precursor ion with a m / z value of 845.9529 , corresponding 
 to a ubiquitinated peptide spanning residues f514k527 of phot1 
 ( theoretical monoisotopic mass value , 845.9542 ; error 
 in the mass observed in the precursor ion , 1.4 ppm ) .
ubiquitinated 
 lysine is shown in the sequence as k , as it is labeled 
 by the digly tryptic remnant of ubiquitin .
thereafter , one - third of the samples 
 were collected immediately under dim safe light ( dark ) , another third 
 were irradiated with blue light for 20 min ( blue ) , and the remaining 
 seedlings were incubated in the dark for 60 min after 20 min of blue 
 light irradiation ( recovery ) .
microsomal proteins were extracted from 
 these seedlings and immunoprecipitated with an anti - gfp antibody . 

the microsomal proteins ( input ) and the immunoprecipitates ( ip ; anti - gfp ) 
 were resolved by a 7.5% sds - page gel , transferred to a nitrocellulose 
 membrane , stained by deep purple fluorescent stain ( a ) , and detected 
 by an anti - ubiquitin antibody ( b ) . in panel a , phot1gfp with 
 lower and higher electrophoretic mobilities is marked with stars . 
 in panel b , ubiquitination of phot1
crop yield is , to a large extent , dependent on the photosynthetic 
 activity of the green leaves of the developing plant .
recent studies 
 have shown that the photosynthetic efficiency of these organs is optimized 
 via a number of blue light / uv - a - mediated responses , such as phototropism 
 in the upper region of the plant , chloroplast movement , stomatal opening , 
 palisade cell development , and leaf flattening . in 
 the model organism a. thaliana ,
all 
 of these physiological processes are mediated by phototropins ( phot1 
 and phot2 ) , as reviewed by christie et al .
however , the signaling cascade that leads from the photoexcitation 
 of phot1 ( or phot2 ) to the corresponding physiological response has 
 not yet been elucidated in detail .
the aim of this study was to explore 
 this signaling pathway in arabidopsis further .
we detected the phosphorylation of phot1 and accumulation 
 of web1 in the membrane - associated protein fraction by 2d dige ( figures 24 ) , and we identified 
 a large number of phot1 ser / thr sites phosphorylated in vivo ( figure 5 ) as well as a phot1 ubiquitination site , which 
 was identified by mass spectrometry analysis of phot1gfp co - immunoprecipitates 
 ( figure 7 ) .
these results further revealed 
 the complexity of phot1 post - translational modifications . since phototropins were first found to be membrane - associated proteins 
 that are phosphorylated upon blue light irradiation in etiolated seedlings 
 of pea ( pisum sativum ) , much progress has been made in characterizing 
 the phosphorylation of phototropins .
salomon et al . identified eight ser residues in oat phot1a in vitro that 
 are phosphorylated , which are hierarchically located either in the 
 n - terminus or hinge 1 region .
sullivan et al . identified four phosphorylation sites in vivo in arabidopsis phot1 : two in the n - terminus and two 
 in the hinge 1 region .
inoue et al . identified 
 eight ser / thr residues in arabidopsis phot1 , including one in the kinase domain and one in the c - terminus 
 ( that we did not detect ) in addition to six in the n - terminus or hinge 
 1 region .
interestingly , all the four sites identified by sullivan 
 et al . were observed by inoue et al .
however , the large number of individual protein 
 spots ( over 50 ) from the two rows of phot1 spanning from the basic 
 to the acidic regions detected in our 2d dige gels ( figure 2a ) indicates the greater complexity of phot1 phosphorylation . 

our study identified 14 discrete ser / thr sites phosphorylated in vivo , 
 which included six sites identified by sullivan et al . and inoue et al .
in addition , we identified eight 
 additional ser / thr sites phosphorylated in vivo from unirradiated 
 and blue light - exposed samples .
a string of parallel phot1 spots was 
 detected in the unirradiated etiolated samples ( figure 2a , c ) , and they were shifted to more basic regions after phosphatase 
 treatment , indicating that some sites of phot1 were phosphorylated 
 even in the dark . in this study , we identified three such phosphoserine 
 residues from phot1 in seedlings without blue light treatment ( figure 5 ) . despite the large number of phosphorylation sites identified for 
 phototropins in this and other studies ,
kinoshita et al . reported the binding 
 of a 14 - 3 - 3 protein to s358 in vicia faba phot1a and s344 in v. faba phot1b 
 and proposed that the binding is likely a key step in the phototropin - mediated 
 stomatal opening response .
report the binding of a 14 - 3 - 3 protein to s350 and s376 of arabidopsis phot1 ; because s376 corresponds to s344 
 in v. faba phot1b , it could possibly serve the same role .
inoue et al . showed 
 that autophosphorylation of s851 in the kinase activation loop was 
 essential for a whole series of phot1-mediated responses in arabidopsis and proposed a possible secondary role 
 for s849 .
finally , tseng et al . demonstrated 
 that s747 in arabidopsis phot2 bound 
 the  isoform of a 14 - 3 - 3 protein in a yeast two - hybrid screen 
 and that mutating that serine to alanine blocked phot2-mediated stomatal 
 opening .
the mutation failed to have 
 an impact on phototropism , however , indicating a high level of specificity . 
 sorting out the roles of the many phototropin phosphorylations remains 
 a daunting task .
a string of protein spots was observed in the phot1 region in the 
 microsomal protein samples from either the unirradiated or irradiated 
 seedlings , even after overnight phosphatase treatment of the microsomal 
 proteins , suggesting additional post - translational modifications other 
 than phosphorylation and/or incomplete phosphatase treatment . in additional 
 to protein phosphorylation
, ubiquitination is another important post - translational 
 modification that regulates protein activity , including protein degradation , 
 membrane protein endocytosis , and subcellular protein trafficking .
showed that phot1 could 
 be mono- , multi- , or polyubiquitinated , depending on light intensity , 
 and phot1-interacting protein nonphototropic hypocotyl 3 ( nph3 ) functions 
 as a substrate adaptor in an e3 cullin3-based ubiquitin ligase .
they 
 also showed that their high - intensity light treatment failed to induce 
 any detectable ubiquitination in vivo in an nph3 mutant , 
 indicating that nph3 is involved in the ubiquitination that we detected .
in addition , we found that ubiquitination of phot1 followed a pattern 
 similar to that for phosphorylation : both increased after blue light 
 exposure but subsequently decreased during the recovery stage following 
 a saturating light exposure .
it seems likely that ubiquitination in 
 addition to phosphorylation could account for the increase in mass 
 that we detected for phot1 on illumination in vivo .
lys526 is very 
 close to the attachment of the j- helix to the lov2 domain , 
 so it is reasonable to expect that it could affect lov domain unfolding 
 following photoexcitation or folding during dark recovery . in this 
 manner
a tryptophan , w491 , is also extremely close to lys526 , 
 and hoersch et al .
presented evidence 
 that this tryptophan is moved into a more hydrophilic environment 
 upon lov domain photoexcitation , indicative of the protein conformational 
 changes in this region of the molecule .
because they are unstable , of low abundance , and often in low stoichiometry , 
 phosphopeptides are hard to detect by mass spectrometry alone , and 
 immunoprecipitation or other methods are often used to enrich specific 
 proteins to identify phosphopeptides .
even so , only a small number 
 of phosphopeptides has been previously identified for phot1 .
however , 2d dige , which separates two or three samples in the same 
 gel , can detect slight differences in pi as a result of a change in 
 phosphorylation at any residue of the protein .
therefore , the shifted 
 spots in 2d dige provide a more accurate estimate of protein phosphorylation 
 sites . as shown before , 2-de gels resolved 
 over 20 spots of the brassinosteroid - signaling transcription factor 
 bzr1 , which is consistent with the number of phosphorylation sites 
 predicted on the basis of the consensus substrate sequence for the 
 bin2/gsk3 kinase . only with the combination 
 of protein fractionation and enrichment , tandem mass spectrometry , 
 and 2d dige can a more accurate picture of specific protein phosphorylation 
 and dephosphorylation be elucidated .
our study and previous studies showed that more than 16 phosphorylation 
 sites and a number of other post - translationally modified residues 
 affecting charge are likely present for phot1 .
further studies will 
 be required to characterize the biochemical complexity of the phot1 
 protein and its physiological implications . except for the phosphorylation 
 sites in the kinase domain ,
the function of other phosphorylation sites is not known for phototropin - mediated 
 signaling , but it might be expected to play some role in one or more 
 blue light - induced physiological responses .
 plants adapt to environmental light conditions by photoreceptor - mediated 
 physiological responses , but the mechanism by which photoreceptors 
 perceive and transduce the signals is still unresolved . here , we used 
 2d difference gel electrophoresis ( 2d dige ) and mass spectrometry 
 to characterize early molecular events induced by short blue light 
 exposures in etiolated arabidopsis seedlings . 
 
 we observed the phosphorylation of phototropin 1 ( phot1 ) and accumulation 
 of weak chloroplast movement under blue light 1 ( web1 ) in the membrane 
 fraction after blue light irradiation . over 50 spots 
 could be observed 
 for the two rows of phot1 spots in the 2-de gels , and eight novel 
 phosphorylated ser / thr sites were identified in the n - terminus and 
 hinge 1 regions of phot1 in vivo . 
 blue light caused ubiquitination 
 of phot1 , and k526 of phot1 was identified as a putative ubiquitination 
 site . 
 our study indicates that post - translational modification of 
 phot1 is more complex than previously reported . 
Introduction
Materials and Methods
Results
Discussion