cyanobacterial lineages also contain a recognizable csk homolog , confirming the evolutionary origin of this chloroplast protein from cyanobacteria .
1 ) , consistent with the secondary symbiotic origin of diatom and phaeophycean plastids from red algae . interestingly , a csk ortholog is also found in  chromatophoresin this context , cyanobacterial endosymbionts  of the amoeboid eukaryote paulinella chromatophora .
the symbiotic origin of paulinella chromatophores has an independent evolutionary history from the symbiotic event that gave rise to chloroplasts ( nowack et al .
csk is present in all major plant and algal lineages and evolved from a cyanobacterial sensor histidine kinase .
bayesian posterior probabilities are shown above nodes , phyml 3.0 bootstrap values are shown below nodes .
the name of each taxon is colored according to the major photosynthetic pigment characteristic of that group . the phylogenetic analysis presented in figure 1 and table 1
csk occurs as a canonical sensor histidine kinase in cyanobacteria , red algae , diatoms , and phaeophytes ( table 1 ) , whereas in green algae and plants , csk is a modified histidine kinase as the conserved histidine autophosphorylation site in csk has been lost in these lineages .
studies in cyanobacteria suggest that the narl - type response regulator ycf29 is the cognate partner of csk ( sato et al .
csk seems to retain its cognate response regulator partner in cyanobacteria and in nongreen algae ( table 1 ) but not in green algae and plants  lineages in which csk occurs as a modified histidine kinase ( table 1 ) .
the evolutionary loss of a bacterial - type response regulator from chloroplasts may therefore correlate with a modified kinase activity of csk ( puthiyaveetil and allen 2009 ) .
distribution of csk , ycf29 , and ptk in photosynthetic organisms note.the plus ( + ) indicates the presence and the minus (  ) indicates the absence of csk or ycf29 or ptk . the abbreviation  nk
 indicates that the complete genome sequence for that taxon is not available , so the presence or absence of csk or ycf29 or ptk is not known . the subcategories 
n  and  p  in the csk , ycf29 , and ptk occurrence column stand for  nuclear  and  plastid ,  respectively , and indicate the genetic compartment in which these proteins are encoded . for p. chromatophora ,
the accession numbers of c. punctiforme pcc 73102 csk and ycf29 homologs are acc82407 and acc80206 , respectively ; for c .sp .
the taxonomic group  viridiplantae  means  green plants ,  and includes green algae , lower , and higher plants .
in order to investigate protein  protein interactions between csk and its putative functional partners , we undertook a yeast two - hybrid analysis .
figure 2a shows growth of all bait  prey combinations in a medium lacking leucine and tryptophan , confirming the successful transformation of yeast cells with both bait and prey plasmids .
figure 2b shows growth of yeast cells in a medium lacking leucine , tryptophan , and histidine .
growth on this latter medium reports on interactions of bait and prey proteins , which together activate the his reporter gene , enabling yeast cells to grow in a medium lacking histidine .
figure 2b shows that functional interactions occurred between the following pairs of bait and prey proteins : csk with csk ; csk with sig-1 ; ptk with csk ; ptk with sig-1 ; ferredoxin - nadp reductase ( fnr ) with ia2 .
fnr and ia2 are two chloroplast proteins that are known to interact ( kuchler et al .
2002 ) and are therefore used as a positive control as in juric et al .
prey combination fnr/220 is used as a negative control as these chloroplast proteins do not interact ( juric et al . 2009 ) and are thus unable to permit growth in a medium lacking histidine ( fig .
the combinations csk / pbd and ptk / pbd are additional negative controls designed to reveal self - activation of the bait proteins  csk and ptk .
fnr/220 , csk / pbd , and ptk / pbd are negative controls . test combination of bait and prey proteins are csk / csk , csk / sig-1 , csk / tcp34 , ptk / csk , ptk / sig-1 , and ptk / tcp34 .
( a ) yeast cells growing on synthetic sd media plates lacking leucine and tryptophan .
the growth of all bait and prey combinations in this medium confirms successful transformation of yeast cells with both bait and prey plasmids .
( b ) growth of yeast cells in sd media plates without leucine , tryptophan , and histidine .
the growth in his plates require the activation of the histidine biosynthetic gene ( his reporter gene ) , which in turn requires the functional interaction between bait and prey proteins .
protein interactions inferred from the results in figure 2 by use of a second reporter gene , -galactosidase .
this gene is under the control of a separate promoter from that of the his reporter gene , and its activation is therefore an independent measure of interaction .
the results from the -galactosidase assay shown in figure 3 are also indicative of the relative strength of the different protein  protein interactions . among the test combinations
, csk / csk shows the highest degree of interaction ; followed by csk / ptk , with csk / sig-1 and ptk / sig-1 showing weaker and more or less equal interactions ( fig .
the results in figures 2 and 3 suggest clearly that tcp34 does not interact with csk or ptk .
our results therefore do not support the response regulator function of tcp34 in plant chloroplasts .
activation of the -galactosidase reporter gene ( lacz gene ) , which is driven by a separate promoter from the his reporter gene , requires functional interaction between bait and prey proteins .
-galactosidase activity is also a measure of the strength of interaction , with higher activity suggesting stronger interaction .
one -gal unit hydrolyses 1 mol of o - nitrophenyl--d - galactopyranoside ( onpg ) to o - nitrophenol and galactose per minute at ph 7.5 and 37 c .
error bars indicate the standard error of the mean of 3 individual measurements , with each measurement representing a different sample .
arabidopsis knockout mutants of the csk gene are unable to repress photosystem i genes in photosystem i light and therefore can not regulate the relative quantities of photosystem i and photosystem ii ( puthiyaveetil et al . 2008 ) .
sigma factors assist rna polymerase in promoter recognition and dna - melting , two processes that determine faithful and efficient transcription ( wosten 1998 ) .
as many as six sigma factors are found in arabidopsis chloroplasts ( lysenko 2007 ) .
sig-1 has been shown to be phosphorylated under pq oxidizing conditions , when incident light favors photosystem i ( light 1 ) ( shimizu et al . 2010 ) . phosphorylated sig-1 represses transcription at psa ( photosystem i reaction center ) promoters while efficiently transcribing psb ( photosystem ii ) promoters ( shimizu et al . 2010 ) .
sig-1 phosphorylation is part of the control of gene expression involved in photosystem stoichiometry adjustments , but the identity of the sig-1 kinase is yet to be revealed ( shimizu et al .
first , csk function and sig-1 phosphorylation have the same gene expression phenotype , which is suppression of psa genes ( puthiyaveetil et al .
this identical gene - regulatory property of these two proteins suggests they are part of the same signal transduction pathway .
secondly , csk and sig-1 interact in vivo in yeast ( figs . 2 and 3 ) .
thirdly , oxidized pq , the signal for sigma factor phosphorylation , is also the signal that promotes the kinase activity of csk ( ibrahim i m , puthiyaveetil s , allen jf , unpublished data ) . the high degree of interaction between csk monomers , as noted in our yeast two - hybrid assay ( figs . 2 and 3 ) , suggests that csk exists as a homodimer in its functional form .
this property of csk is consistent with the proposed signal sensing function , as dimerization is well known in bacterial sensor kinases ( wolanin et al .
ptk is a eukaryotic serine / threonine protein kinase of the casein kinase ii family ( baginsky et al .
ptk is usually found associated with the plastid - encoded rna polymerase ( pep ) , acting as a global regulator of chloroplast transcription ( link 2003 ) . in low light conditions
, ptk keeps chloroplast transcription at a low level by phosphorylating pep  phosphorylated pep transcribes chloroplast genes less effectively than unphosphorylated pep ( baginsky et al .
a single subunit of pep  the 72 kda  ' subunit  is usually found in its phosphorylated form in low light ( baginsky et al .
ptk from mustard ( sinapis alba l. ) can also phosphorylate sig-1 in vitro ( ogrzewalla et al . 2002 ) .
these observations , taken together with our yeast two - hybrid results ( figs . 2 and 3 ) , suggest that ptk nonspecifically suppresses chloroplast transcription in low light by phosphorylating pep structural (  ' subunit ) and regulatory ( sigma factor ) subunits .
light 1 ( photosystem i light ) and light 2 ( photosystem ii light ) are selective for electron transport through photosystem i or photosystem ii only when light intensity is rate limiting for photosynthesis , and other factors , such as co2 concentration or temperature , are not . for selective transcriptional control of reaction center gene transcription in arabidopsis thaliana ,
conditions correspond to photon flux densities of the order of 12 e m s ( puthiyaveetil and allen 2008 ) . because ptk - mediated suppression of chloroplast transcription occurs at low incident light intensity ( baena - gonzalez et al .
2001 ) , it is interesting to ask how might specific reaction center gene transcription be achieved in light 1 and 2 ?
we suggest that , under these conditions , the activity of ptk is overridden by two regulatory proteins 
4a and b ) . the proposed chloroplast signal transduction pathway coupling the redox state of the photosynthetic electron carrier plastoquinone in the chloroplast thylakoid membrane with initiation of transcription of chloroplast dna at the promoter regions of the genes psa ( encoding reaction center proteins of photosystem i ) and psb ( encoding reaction center proteins of photosystem ii ) .
( a ) under light 1 , a rate - limiting low light selective for photosystem i , electron flow through photosystem i ( ps i ) has a greater potential than that through photosystem ii ( ps ii ) , and so pq pool is maintained in its oxidized form .
csk is autophosphorylated and active as a protein kinase using both sig-1 and ptk as substrates : sig-1 and ptk are thus maintained in their phosphorylated forms .
phospho - sig-1 represses transcription at the psa promoter while allowing transcription of psb genes .
phospho - ptk is inactive ; therefore , it can not suppress chloroplast transcription nonspecifically , and under this circumstance , only csk - mediated  via phospho - sig-1specific repression of psa genes occurs .
this differential reaction center gene transcription increases the stoichiometry of photosystem ii relative to photosystem i. ( b ) under light 2 , a rate - limiting low light selective for photosystem ii , electron flow through photosystem i ( ps i ) has a lower potential than that through photosystem ii ( ps ii ) , and so pq pool is maintained in its reduced form ( pqh2 ) .
the repression of psa genes , occurred during light 1 , is now relieved by the action of a pep phosphatase that catalyzes dephosphorylation of phospho - sig-1 . as a result , ps i transcription increases . under this light condition
however , the action of pep phosphatase overrides ptk activity by dephosphorylating both sig-1 as well as pep subunits so that nonspecific repression of reaction center genes is counteracted .
the increase in photosystem i transcription in light 2 , therefore , leads to an increase in photosystem i units relative to photosystem ii .
we thus propose that in light 1 , in order to bring about specific down - regulation of psa genes , ptk becomes inhibited ( fig .
phosphorylation inhibits ptk ( link 2003 ) , and yet no ptk kinase has previously been identified . in the light of the strong interaction between csk and ptk found in our yeast two - hybrid assay ( figs . 2 and 3 ) , we here propose that csk acts as the ptk kinase , catalyzing phosphorylation and inactivation of ptk .
csk itself then catalyzes phosphorylation of sig-1 , which has the effect of repressing only psa transcription . as discussed earlier ,
psb transcription is unaffected by sig-1 phosphorylation , thus the quantity of photosystem ii increases in light 1 relative to photosystem i ( fig .
, however , when photosystem i is rate limiting , sigma factors are predicted to become dephosphorylated to relieve the repression on psa genes ( fig .
the phospho - pep phosphatase can perform both of these functions by dephosphorylating sig-1 as well as the pep structural subunits ( fig .
4b ) . whether such a phospho - pep phosphatase is constitutively active or regulated by redox signals remains to be seen .
we suggest that ptk , a eukaryotic - type protein kinase , displaced an analogous prokaryotic - type response regulator as the functional partner of csk in a two - component regulatory system .
this proposal is supported by the distribution of csk , ycf29 , and ptk between different eukaryotic lineages ( table 1 ) .
ptk seems to be present in lineages that have lost ycf29 , thus supporting the notion of ptk replacing ycf29 as the functional partner of csk .
we conclude that regulation of photosystem stoichiometry in chloroplasts ( allen 1995 , 2005 ; pfannschmidt , nilsson , and allen 1999 ) results from effects of the redox state of the plastoquinone pool on reversible phosphorylation of csk , ptk , and sig-1 .
these proteins interact with each other and together comprise the redox signal transduction pathway that acts to regulate transcription of chloroplast reaction center genes in response to imbalance in rates of energy conversion in photosystems i and ii ( pfannschmidt , nilsson , and allen 1999 ; pfannschmidt , nilsson , tullberg , et al .
this genetic switch has been retained from the cyanobacterial ancestor of chloroplasts ( puthiyaveetil and allen 2009 ) and is a transcriptional analog of posttranslational modification of the activity of photosystem i and ii by phosphorylation of chloroplast light - harvesting complex ii ( allen et al . 1981 ; allen 1992 ) .
redox control of transcription ( allen 1993c ) has been proposed as a necessary condition for the retention , in evolution , of the genetic systems of chloroplasts and mitochondria as extranuclear , cytoplasmic elements that produce non - mendelian inheritance of characters associated with photosynthesis and respiration in eukaryotic cells ( allen 1993a , 1993b ; race et al .
the results described here are in agreement with corr and further resolve the mechanism by which a modified bacterial two - component system continues to operate in chloroplasts in order to secure a functionally intelligible adjustment of the stoichiometry of the chloroplast photosystems in response to changing light regimes .
multiple alignment of the amino acid sequence corresponding to the catalytic domain of csk and its homologs was generated across a representative selection of photosynthetic eukaryotes and cyanobacteria using clustalx and adjusted manually using jalview ( clamp et al .
bayes 3.1 ( ronquist and huelsenbeck 2003 ) from 2,000,000 generations divided between two parallel runs of 1,000,000 , each with sampling every 1,000 generations .
the substitution model was inferred using a mixed model of amino acid substitution , and rate across sites variation was modeled on a discrete gamma distribution approximated using 4 gamma categories and 1 category of invariable sites .
bootstraps were generated using phyml 3.0 ( guindon and gascuel 2003 ) using the whelan and goldman substitution model and rate across sites variation modeled on an approximate gamma distribution using 4 gamma categories and one category of invariable sites .
the yeast two - hybrid assay employed in our study is based on the gal4 system ( stratagene ) .
bait proteins were fused to the activation domain ( ad ) and prey proteins to the binding domain ( bd ) .
cdnas encoding bait proteins are therefore cloned into the pad - gal4 - 2.1 vector and prey cdnas into the pbd - gal4 vector .
a cdna fragment encoding the kinase domain ( q301-a611 ) of arabidopsis csk ( product of gene at1g67840 ) was amplified ( for primers , see supplementary table 1 , supplementary material online ) from a full - length csk cdna clone used in an earlier study ( puthiyaveetil et al .
this was then used as a bait protein . in order to study whether csk forms dimers , the same cdna region of csk
the other prey proteins tested as candidates of csk s partner were sig-1 ( product of gene at1g64860 ) and tcp34 ( product of gene at3g26580 ) .
for sig-1 , the cdna region encoding the mature arabidopsis protein ( a81-n502 ) was amplified ( primers , supplementary table 1 , supplementary material online ) from the full - length arabidopsis biological resource centre ( abrc ) clone u16526 . for tcp34 , the cdna region encoding the mature arabidopsis protein  without the c - terminal transmembrane anchor(l28-g324 )
was amplified ( primers , supplementary table 1 , supplementary material online ) from the abrc clone u24715 .
the cdna region encoding the mature ptk protein ( product of gene at2g23070 ) was amplified ( primers , supplementary table 1 , supplementary material online ) from the abrc clone u15758 .
the prey proteins tested against the ptk bait were csk ; sig-1 ; and tcp34 .
the cdna regions cloned for these prey proteins were the same as those described above . in a negative control ,
the complete fnr protein was used as the bait against the  220  domain of the trol protein , essentially as described ( juric et al .
csk and ptk bait proteins were tested for self - activation by using bd  encoded by the pbd - gal4 vector  as prey proteins .
ia2  domain of tic62 protein as prey , as described previously ( juric et al . 2009 ) .
all bait and prey plasmid combinations were transformed using liac method into the yeast strain saccharomyces cerevisiae yf53 ( mata ura352 his3200 ade2101 lys2801 trp1901 leu23,112 gal4542 gal80538 , with his3 and lacz reporter gene constructs lys2::uasgal1tatagal1his3 and ura3::uasgal4 17 mers ( x3)tatacyc1lacz ) .
yeast transformants were selected on synthetic dropout ( sd ) media without leucine and tryptophan .
protein interactions were detected by growth on sd media lacking leucine , tryptophan , and histidine and by the -galactosidase assay .
all assays were performed in duplicates from three independent colonies in 0.5 ml of reaction buffer , and -galactosidase units were calculated as described ( feilotter et al .
multiple alignment of the amino acid sequence corresponding to the catalytic domain of csk and its homologs was generated across a representative selection of photosynthetic eukaryotes and cyanobacteria using clustalx and adjusted manually using jalview ( clamp et al .
bayes 3.1 ( ronquist and huelsenbeck 2003 ) from 2,000,000 generations divided between two parallel runs of 1,000,000 , each with sampling every 1,000 generations .
the substitution model was inferred using a mixed model of amino acid substitution , and rate across sites variation was modeled on a discrete gamma distribution approximated using 4 gamma categories and 1 category of invariable sites .
bootstraps were generated using phyml 3.0 ( guindon and gascuel 2003 ) using the whelan and goldman substitution model and rate across sites variation modeled on an approximate gamma distribution using 4 gamma categories and one category of invariable sites .
the yeast two - hybrid assay employed in our study is based on the gal4 system ( stratagene ) .
bait proteins were fused to the activation domain ( ad ) and prey proteins to the binding domain ( bd ) .
cdnas encoding bait proteins are therefore cloned into the pad - gal4 - 2.1 vector and prey cdnas into the pbd - gal4 vector .
a cdna fragment encoding the kinase domain ( q301-a611 ) of arabidopsis csk ( product of gene at1g67840 ) was amplified ( for primers , see supplementary table 1 , supplementary material online ) from a full - length csk cdna clone used in an earlier study ( puthiyaveetil et al .
this was then used as a bait protein . in order to study whether csk forms dimers , the same cdna region of csk
the other prey proteins tested as candidates of csk s partner were sig-1 ( product of gene at1g64860 ) and tcp34 ( product of gene at3g26580 ) .
for sig-1 , the cdna region encoding the mature arabidopsis protein ( a81-n502 ) was amplified ( primers , supplementary table 1 , supplementary material online ) from the full - length arabidopsis biological resource centre ( abrc ) clone u16526 . for tcp34 , the cdna region encoding the mature arabidopsis protein  without the c - terminal transmembrane anchor(l28-g324 )
was amplified ( primers , supplementary table 1 , supplementary material online ) from the abrc clone u24715 .
the cdna region encoding the mature ptk protein ( product of gene at2g23070 ) was amplified ( primers , supplementary table 1 , supplementary material online ) from the abrc clone u15758 .
the prey proteins tested against the ptk bait were csk ; sig-1 ; and tcp34 .
the cdna regions cloned for these prey proteins were the same as those described above . in a negative control ,
the complete fnr protein was used as the bait against the  220  domain of the trol protein , essentially as described ( juric et al .
csk and ptk bait proteins were tested for self - activation by using bd  encoded by the pbd - gal4 vector  as prey proteins .
the positive control uses the complete fnr protein as bait and the  ia2  domain of tic62 protein as prey , as described previously ( juric et al .
all bait and prey plasmid combinations were transformed using liac method into the yeast strain saccharomyces cerevisiae yf53 ( mata ura352 his3200 ade2101 lys2801 trp1901 leu23,112 gal4542 gal80538 , with his3 and lacz reporter gene constructs lys2::uasgal1tatagal1his3 and ura3::uasgal4 17 mers ( x3)tatacyc1lacz ) .
yeast transformants were selected on synthetic dropout ( sd ) media without leucine and tryptophan .
protein interactions were detected by growth on sd media lacking leucine , tryptophan , and histidine and by the -galactosidase assay .
all assays were performed in duplicates from three independent colonies in 0.5 ml of reaction buffer , and -galactosidase units were calculated as described ( feilotter et al .
 chloroplast sensor kinase ( csk ) is a bacterial - type sensor histidine kinase found in chloroplasts  
 photosynthetic plastids  in eukaryotic plants and algae . using a yeast two - hybrid screen , we demonstrate recognition and interactions between : csk , plastid transcription kinase ( ptk ) , and a bacterial - type rna polymerase sigma factor-1 ( sig-1 ) . 
 csk interacts with itself , with sig-1 , and with ptk . 
 ptk also interacts directly with sig-1 . 
 ptk has previously been shown to catalyze phosphorylation of plastid - encoded rna polymerase ( pep ) , suppressing plastid transcription nonspecifically . 
 phospho - ptk is inactive as a pep kinase . here 
 , we propose that phospho - csk acts as a ptk kinase , releasing ptk repression of chloroplast transcription , while csk also acts as a sig-1 kinase , blocking transcription specifically at the gene promoter of chloroplast photosystem i. oxidation of the photosynthetic electron carrier plastoquinone triggers phosphorylation of csk , inducing chloroplast photosystem ii while suppressing photosystem i. csk places photosystem gene transcription under the control of photosynthetic electron transport . 
 this redox signaling pathway has its origin in cyanobacteria , photosynthetic prokaryotes from which chloroplasts evolved . 
 the persistence of this mechanism in cytoplasmic organelles of photosynthetic eukaryotes is in precise agreement with the corr hypothesis for the function of organellar genomes : the plastid genome and its primary gene products are co - located for redox regulation . 
 genes are retained in plastids primarily in order for their expression to be subject to this rapid and robust redox regulatory transcriptional control mechanism , whereas plastid genes also encode genetic system components , such as some ribosomal proteins and rnas , that exist in order to support this primary , redox regulatory control of photosynthesis genes . 
 plastid genome function permits adaptation of the photosynthetic apparatus to changing environmental conditions of light quantity and quality . 