BACKGROUND
research has shown us that the functions of proteins, including metabolic pathway enzymes, are highly regulated through compartmentalization, covalent modifications and specific protein-protein or protein-small molecule interactions.
most protein-protein interactions are mediated through specific domains that recognize other domains, motifs or modifications on other proteins  <cit> .
this has lead to the compilation of a host of recognizable domains that bind other protein domains or other molecules, such as phosphoinositides, nucleic acids or carbohydrates.
these are often easily detected through bioinformatic methods and provide clues about protein function.
among these domains are a collection of well characterized carbohydrate docking domains  <cit>  one of which is present on the starch dephosphorylating enzyme sex <dig>  sex <dig> belongs to the protein tyrosine phosphatase superfamily of protein phosphatases  <cit>  and has been shown to be plastid localized, docks onto starch and is the functional equivalent of the homo sapiens laforin phosphatase that dephosphorylates glycogen  <cit> .
during our original analysis of the carbohydrate binding domain of sex <dig> we noted several other arabidopsis proteins with the equivalent carbohydrate docking domain, including at5g <dig>  <cit> .
at5g <dig> is a small protein that has all the predicted amino acids necessary to bind starch.
here we have performed a biochemical and informatics analysis of at5g <dig> and show that like sex <dig>  at5g <dig> is highly conserved across plant species, is plastid localized, binds starch and is coordinately regulated at both the protein and message level along with other associated carbohydrate binding proteins.
we have also noted that at5g <dig> has a well-conserved predicted coiled-coil domain, like a number of the coordinately expressed starch metabolic enzymes and may function as protein interaction scaffold on the starch granule.
this is consistent with recent genetic and biochemical data indicating that many of the starch metabolic enzymes interact physically  <cit> .
RESULTS
bioinformatic analysis of at5g <dig> predicts protein structural features, subcellular localization and gene expression patterns
the amino acid sequence encoded by gene at5g <dig> is presented along with the homologues we identified, in the multiple sequence alignment of figure  <dig>  despite the presence of a few incomplete sequences, it is apparent by inspection that the carboxy-terminal region of the alignment is well conserved.
a previous publication from our laboratory  <cit>  showed the dual-specificity phosphatase sex <dig>  encoded by the gene at3g <dig>  contains a c-terminal carbohydrate-binding domain , which is shared with at5g <dig> .
this domain begins at residue  <dig> of at5g <dig> .
several residues were observed to be conserved in our previous work are also conserved in this alignment ; hciliaris ; rraphanist ; gmax ; ptrichocar ; cclementin ; csinensis ; toficinale ; osativa ; hvulgare ; graimondii ; mtruncatul ; pglauca ; ptaeda ; creinhardt .
structural domains were defined as detailed in methods.
both the strongly predicted coiled-coil  domain and the carbohydrate-binding domain  are indicated by underlined bars.
the region immediately preceding the cbm is also well conserved in the alignment.
we have analyzed the structural properties of this region as detailed in the methods section.
we found no evidence of characterized protein sequence domains.
however, we found consistent evidence of a coiled-coil domain in all the sequences.
this data is summarized in the additional table  <dig>  there is a pattern of a strongly predicted region, followed by a more weakly predicted region.
the stronger region extends from roughly residue  <dig> to  <dig> in the at5g <dig> sequence .
the weaker region extends from roughly residue  <dig> to  <dig> in the at5g <dig> sequence .
it is evident that prior to about position  <dig> in the alignment, the n-terminal region is much less highly conserved.
we have analyzed this set of sequences for the presence of chloroplast transit peptides, and the data are presented in additional table  <dig>  it has been suggested previously  <cit>  that sequences be judged positive for a chloroplast transit peptide if they achieve three out of four positive predictions in this test regimen.
by that standard, sequence at5g <dig> is predicted to be positive.
by this criterion, one other sequence  is also positive .
three other sequences might be termed "possibly positive" on the basis of a positive result in  <dig> of the  <dig> tests .
the remainder of the sequence set either has only one positive test  or none positive .
the presence of a well-conserved cbm and the presence of a predicted chloroplast transit peptide suggested to us the possibility that the protein product of gene at5g <dig> participates in carbohydrate metabolism.
we investigated this hypothesis further by examining patterns of correlated gene co-expression data as detailed in the methods section.
we examined a set of  <dig> genes known to be involved in starch metabolism  given in  <cit> ).
we found that eight of these have correlation coefficients greater than r =  <dig>    <dig> ; at5g <dig>   <dig> ; at3g <dig>   <dig> ; at1g <dig>   <dig> ; at4g <dig>   <dig> ; at1g <dig>   <dig> ; at1g <dig>   <dig> ; at1g <dig>   <dig> ).
we next examined the patterns of expression for at5g <dig> under various experimental conditions at the genevestigator site.
it appeared that expression levels varied in a diurnal pattern.
we have downloaded the original microarray data for the diurnal experiment of smith et al.
<cit> , and have compared the temporal expression patterns of at5g <dig> with the set of eight starch metabolic enzyme genes cited above whose general co-expression pattern are most highly correlated as revealed at the arabidopsis co-expression tool site.
the results are shown in figure  <dig>  it is readily apparent that at5g <dig> and these starch metabolic enzymes show a similar overall diurnal pattern, with expression declining during the first half of the experiment , and rising to a maximum near the end of the experiment .
the correlation coefficients between the expression pattern of at5g <dig> and each of these genes are as follows: at1g <dig>   <dig> ; at2g <dig>   <dig> ; at1g <dig>   <dig> ; at3g <dig>   <dig> ; at1g <dig>   <dig> ; at1g <dig>   <dig> ; at5g <dig>   <dig> ;at4g <dig>   <dig> .
the mean correlation coefficient for this set of data is r =  <dig> .
the presence of a strongly predicted coiled-coil  domain in the amino acid sequence of at5g <dig>  suggested to us the possibility of protein-protein interactions.
we therefore screened the reference set of starch metabolic proteins for predictions of chloroplast transit peptides  and cc domains.
<dig> of the  <dig> sequences proved to be negative for cc domains by all three tests.
the other  <dig> had various cc regions predicted, at different strengths, in the different methods.
the evidence for the subset of  <dig> proteins with the strongest cc predictions is presented in additional table  <dig> and the full set of predictions is summarized in additional table  <dig>  there is at least moderate support by one prediction method, and reinforcement by a second method, for cc domains in five starch synthetic enzymes , at1g <dig> , at3g <dig> , at1g <dig> , at4g <dig> ) and two starch degradative enzymes , at1g <dig> ).
the evidence is most definitive for sts <dig> and sts <dig>  where strong predictions are obtained by all three methods for at least one region each .
sts <dig> is especially notable in that the predicted coiled-coil region is exceptionally long .
of the  <dig> starch metabolic proteins set,  <dig> failed to achieve a consensus positive chloroplast transit peptide prediction .
all of these failures have been confirmed to be plastidic by experiment  <cit> .
in order to provide context for the general gene co-expression results with at5g <dig>  we used the arabidopsis coexpression data mining tools site to obtain the correlation coefficients for all-against-all comparisons of the  <dig> genes in the starch metabolic proteins set, over the full nasc microarray database.
we set an arbitrary cutoff of r =  <dig>  and above to define the strongest class of gene co-expression correlations.
we found, as one would expect, a pattern of highly correlated co-expression between genes encoding starch synthetic enzymes, and also between genes encoding starch degradative enzymes.
however, we also observed a number of instances of unexpected high correlation between starch synthetic and starch degradative enzymes.
the correlated gene groupings are depicted in additional table  <dig>  the numerical values for all the gene co-expression correlation comparisons are presented in additional table  <dig>  the most remarkable standout in this collection of data is at4g <dig> .
it has strongly correlated co-expression with the largest number of other starch metabolic enzyme genes , and strong correlations with the largest number of starch degradative enzyme genes .
carbohydrate binding preference of at5g39790
in order to test that at5g <dig> is a carbohydrate binding protein, as predicted by  <cit>  , a carbohydrate binding experiment was performed with at5g <dig> cloned, expressed and purified without the putative chloroplast transit peptide .
the protein was incubated with starch, amylopectin or amylose, and was observed in the pellet fractions with all three carbohydrates by western blot analysis, as shown in figure  <dig>  these results confirm the prediction that this gene product is indeed a carbohydrate binding protein.
the predicted molecular weight is close to  <dig> , as expected due to the presence of the prset purification tag .
although the protein bound all three carbohydrates, some unbound protein was observed in the supernatant of the amylopectin sample, indicating that at5g <dig> has a preference for the longer α 1→ <dig> linked glucose units found in amylose rather than the shorter regions found in α 1→ <dig> branched amylopectin.
the binding of at5g <dig> to starch was also studied as a function ph, redox state and magnesium ion concentration.
during the day the stromal ph is more alkaline, and redox conditions more reducing, than at night  <cit> .
as well, stromal magnesium ion concentrations fluctuate between  <dig> mm during the day and  <dig> mm at night  <cit> .
the results of this study, presented in figure  <dig>  show that at5g <dig> does not dissociate from starch as a function of ph, mg2+ concentration, or redox state.
diurnal protein accumulation profile of at5g <dig> is correlated with microarray mrna transcript levels
the low natural abundance of at5g <dig> in a. thaliana leaves made detection of signals on western blots difficult, so the spinach  results are presented here.
our ability to obtain a stronger signal in spinach is unclear and may simply reflect higher expression in spinach for some unknown reason.
results in figure  <dig> show that protein levels increase during the day and decrease at night over the course of  <dig> hours when equal loading of fresh weigh leaf extracts were analyzed.
focusing on the data between time points 12– <dig> h shows a direct correlation between the protein level and the a. thaliana mrna levels determined by microarray analysis .
both fluctuate synchronously during day/night conditions.
in a control western blot experiment using only the secondary antibody, no protein was detected .
earlier research to monitor diurnal changes in starch metabolizing enzymes  <cit>  was also performed using fresh weight leaf extracts for both mrna analysis by microarray and transcript analysis by western blotting.
these researchers showed that many proteins involved in starch biosynthesis had protein levels that stayed constant even when their mrna levels fluctuated diurnally .
it was proposed that protein levels were under post-transcriptional control.
an exception was granule-bound starch synthase, gbs <dig>  a protein embedded in the starch matrix responsible for the synthesis of the amylose fraction of starch .
protein levels for gbs <dig> increased during the day, and decreased at night as the starch granule was degraded  <cit> .
our results with at5g <dig> show a similar pattern, and this is not unexpected given the fact that dissociation from starch as a function of ph, mg2+ concentration, or redox state was not observed.
chloroplast localization of at5g39790
the data presented in additional table  <dig> of this study predicts that at5g <dig> is chloroplast localized.
to test this prediction we performed three experiments.
in the first experiment chloroplasts were isolated from spinach instead of a. thaliana  and probed with antibodies for the presence of at5g <dig>  results in figure  <dig> show that our protein is indeed localized in the chloroplast fraction of spinach isolates and absent in the cytosolic fraction.
the second experiment utilized a c-terminal gfp fusion construct of at5g <dig> that contained the putative chloroplast transit peptide .
plasmids containing the gfp fusion construct or the plastid targeted red fluorescent protein  control  <cit> , were bombarded into onion epidermal cells.
figure  <dig> shows that after  <dig> hours the gfp-at5g <dig> and rfp co-localize to the chloroplast .
thus the presence of the putative transit peptide indeed targets at5g <dig> to this organelle.
the third approach was to generate a transgenic arabidopsis thaliana plant expressing at5g39790-gfp.
this shows a clear localization of the enzyme to chloroplasts as evidenced by chlorophyll fluorescence .
discussion
we have shown by experiment that the protein encoded by arabidopsis gene at5g <dig> is chloroplast-localized, and that it binds starch.
this is consistent with bioinformatic analysis of the amino acid sequence, which predicts a chloroplast transit peptide, and a well-conserved c-terminal carbohydrate-binding domain .
other structural analysis strongly predicts the presence of a coiled-coil domain.
such regions most often consist of a repeating heptad pattern of regularly spaced, alternating hydrophobic and hydrophilic residues, resulting in an alpha-helical supercoil between partner protein interaction surfaces  <cit> .
many examples have been characterized, including motor and other cytoskeletal proteins, chromosomal and membrane organizing proteins, and transcription factors.
the presence of both a starch-binding domain and a coiled-coil domain in the structure of at5g <dig> suggests the hypothesis that this is a scaffolding protein, whose structural properties would allow it to bind to the starch granule while also interacting with other proteins by way of its coiled-coil domain.
analysis of gene expression patterns strongly supports the involvement of at5g <dig> in a coordinately-regulated set of carbohydrate-metabolizing proteins.
the correlation coefficients are substantial for general co-expression across a large number of microarray experiments between at5g <dig> and several starch metabolic proteins.
in particular, there is a clear diurnal pattern of expression which at5g <dig> shares with a set of starch metabolic proteins.
smith et al  <cit>  in their original presentation of this diurnal experiment microarray data commented in particular on the marked co-expression pattern of a group of starch degradative enzyme-encoding genes.
we find that at5g <dig> shares an evident pattern of co-expression with several of these degradative enzyme genes, but also with the gene for the synthetic enzyme sts <dig> .
the role of sts <dig> has not been defined clearly, but is expected to function in starch synthesis.
our structural analysis of a set of starch metabolic enzymes showed appreciable coiled-coil  potential in several of their amino acid sequences.
this was especially notable in the starch synthases.
there is a body of previous work, both genetic and biochemical, showing the importance of multi-protein complexes in starch synthesis  <cit> .
recently, in wheat amyloplasts, homo-dimeric complexes between sbe isoforms have been observed, and hetero-multimeric complexes between ssi or ssii and sbe isoforms  <cit> .
in zea mays amyloplasts, a number of pair-wise interactions were demonstrated between starch synthetic enzymes.
of particular interest are large complexes involving ssii and ssiii.
the latter was shown to interact with ssi in a region  that shares similarity with a number of other ssiiis from various plant species  <cit> .
this region aligns with the portion of the arabidopsis sts <dig>  sequence containing cc regions, and in fact we have found that the zea mays sequence also contains cc potential in this region similar to the arabidopsis protein .
hence cc interactions should be considered likely candidates to explain at least some of the multiple protein-protein interactions known to be occurring in the starch synthetic enzyme system.
in our structural analysis, arabidopsis sts <dig>  was distinctive in having the largest predicted cc region, probably extending more than  <dig> residues.
this indicates a high potential to participate in multiple cc interactions with other proteins.
this might indicate that sts <dig> participates in a large multi-protein assembly.
relatively little is known about the details of sts <dig> function.
however, sts <dig> mutant plants were recently analyzed  <cit>  and it appears that this enzyme controls the number of starch granules in plastids, perhaps by initiating starch granule priming.
the authors suggest that this is likely to occur in a large protein complex.
in the present study we observed a surprising number of instances of correlated gene co-expression between starch synthetic and starch degradative enzyme genes.
this was most marked for at4g <dig> , which has high correlations with a number of starch degradative enzyme genes.
correlations of this type might be expected if starch synthetic enzymes and starch degradative enzymes were working together in a multi-protein complex.
it has recently been shown that a heteromeric complex of stisa <dig> and stisa <dig> isoforms is necessary for the initiation of starch granule synthesis in potato  <cit> .
these authors concluded that it is likely that such a situation also occurs in arabidopsis.
indeed, our gene expression data show a high correlation between the general expression patterns of at4g <dig>  and at1g <dig> , with only a slightly weaker correlation with at2g <dig> .
the presence of a number of other such correlations in our dataset might indicate that there are further, as yet undetected, interactions between starch synthetic and starch degradative enzymes.
arabidopsis sts <dig> would appear to be a promising target for further investigations in this area.
the combination of gene co-expression data, protein sequence structural analysis, and the results of the carbohydrate binding studies presented in this study strongly support the hypothesis that at5g <dig> is a scaffold protein closely involved in starch metabolism .
the high degree of conservation of the carbohydrate-binding domain and coiled-coil regions of the sequences of the homologues collected from other plant species makes it very likely that they share the same function.
however, sequence heterogeneity in the amino-terminal region of this protein set is observed, and chloroplast transit peptide predictions have provided mixed results.
bioinformatic prediction of chloroplast transit peptides is a difficult process, since there is little conservation observed at the primary or secondary sequence level in actual transit peptides  <cit> .
indeed, in this study,  <dig> of  <dig> starch metabolic protein sequences in arabidopsis failed to attain the recommended performance of three out of four positive test predictions.
all of these sequences except one are known to be plastid-localized  <cit> .
on a larger scale, the zybailov study identified  <dig> proteins as plastid-localized with a high degree of confidence, but of these only 86% could be assigned chloroplast transit peptides by the targetp technique.
the plastid localization of the at5g <dig> homologues, which currently cannot be assigned chloroplast transit peptides with confidence, will require experimental investigation.
our western blot and fluorescent imaging results agree with the bioinformatics predictions that at5g <dig> localizes to the chloroplast.
the predicted carbohydrate-binding domain of at5g <dig> has been confirmed with the starch binding studies.
our data suggest that at5g <dig> preferentially docks to amylose.
given the nature of starch it is unclear why we did not see complete binding to amylopectin given that starch is ~25% amylose.
we can propose that there is simply adequate binding sites for the protein if 25% of the starch is amylose.
it is possible that the branching of amylopectin actually hinders at5g <dig> binding and reflects a unique feature of at5g <dig> and this may play a role in defining its in vivo function.
of interest is that this protein does not dissociate from starch as a function of ph, mg2+ concentration, or redox state.
as such it is highly probable that this protein binds to starch under both day and night conditions  <cit> , and functions as a scaffolding protein for other proteins to bind to via the cc domain.
not all starch binding proteins bind starch tightly all the time.
the binding of the dual-specificity protein phosphatase sex <dig>  has been shown, in vitro to be regulated both by ph and redox potential  <cit> .
sex <dig> is found to bind starch during the day but not at night.
in contrast, α-glucan water dikinase  binds at night but not during the day  <cit> .
even more interesting is the observation that granule-bound starch synthase , a protein embedded in the starch matrix  has protein levels which fluctuate diurnally  <cit>  in the same manner as at5g <dig>  gbs <dig> does not have a carbohydrate-binding domain, and based on similar diurnal protein levels and starch localization, it is tempting to speculate that gbs1may be anchored by at5g <dig>  future studies utilizing biochemical and molecular genetics approaches will address this question.
CONCLUSIONS
we have found that the cbm protein at5g <dig> is expressed in a diurnal fashion at both the transcript and protein level.
at5g <dig> is conserved among plant species and outside of the cbm, the protein maintains a conserved coiled-coil domain that may act a as scaffold to bind other proteins.
gene expression analysis shows a pattern that correlates well with the expression other starch metabolic enzymes.
the expressed protein readily docks starch and analysis with an antibody generated against at5g <dig> shows the protein is localized to chloroplasts as is a gfp construct of the protein.

 research has shown us that the functions of proteins, including metabolic pathway enzymes, are highly regulated through compartmentalization, covalent modifications and specific protein-protein or protein-small molecule interactions. most protein-protein interactions are mediated through specific domains that recognize other domains, motifs or modifications on other proteins. this has lead to the compilation of a host of recognizable domains that bind other protein domains or other molecules, such as phosphoinositides, nucleic acids or carbohydrates. these are often easily detected through bioinformatic methods and provide clues about protein function. among these domains are a collection of well characterized carbohydrate docking domains one of which is present on the starch dephosphorylating enzyme sex sex belongs to the protein tyrosine phosphatase superfamily of protein phosphatases and has been shown to be plastid localized, docks onto starch and is the functional equivalent of the homo sapiens laforin phosphatase that dephosphorylates glycogen. during our original analysis of the carbohydrate binding domain of sex we noted several other arabidopsis proteins with the equivalent carbohydrate docking domain, including at5g. at5g is a small protein that has all the predicted amino acids necessary to bind starch. here we have performed a biochemical and informatics analysis of at5g and show that like sex at5g is highly conserved across plant species, is plastid localized, binds starch and is coordinately regulated at both the protein and message level along with other associated carbohydrate binding proteins. we have also noted that at5g has a well-conserved predicted coiled-coil domain, like a number of the coordinately expressed starch metabolic enzymes and may function as protein interaction scaffold on the starch granule. this is consistent with recent genetic and biochemical data indicating that many of the starch metabolic enzymes interact physically.  bioinformatic analysis of at5g predicts protein structural features, subcellular localization and gene expression patterns the amino acid sequence encoded by gene at5g is presented along with the homologues we identified, in the multiple sequence alignment of figure despite the presence of a few incomplete sequences, it is apparent by inspection that the carboxy-terminal region of the alignment is well conserved. a previous publication from our laboratory showed the dual-specificity phosphatase sex encoded by the gene at3g contains a c-terminal carbohydrate-binding domain, which is shared with at5g. this domain begins at residue of at5g. several residues were observed to be conserved in our previous work are also conserved in this alignment; hciliaris; rraphanist; gmax; ptrichocar; cclementin; csinensis; toficinale; osativa; hvulgare; graimondii; mtruncatul; pglauca; ptaeda; creinhardt. structural domains were defined as detailed in methods. both the strongly predicted coiled-coil domain and the carbohydrate-binding domain are indicated by underlined bars. the region immediately preceding the cbm is also well conserved in the alignment. we have analyzed the structural properties of this region as detailed in the methods section. we found no evidence of characterized protein sequence domains. however, we found consistent evidence of a coiled-coil domain in all the sequences. this data is summarized in the additional table there is a pattern of a strongly predicted region, followed by a more weakly predicted region. the stronger region extends from roughly residue to in the at5g sequence. the weaker region extends from roughly residue to in the at5g sequence. it is evident that prior to about position in the alignment, the n-terminal region is much less highly conserved. we have analyzed this set of sequences for the presence of chloroplast transit peptides, and the data are presented in additional table it has been suggested previously that sequences be judged positive for a chloroplast transit peptide if they achieve three out of four positive predictions in this test regimen. by that standard, sequence at5g is predicted to be positive. by this criterion, one other sequence is also positive. three other sequences might be termed "possibly positive" on the basis of a positive result in of the tests. the remainder of the sequence set either has only one positive test or none positive. the presence of a well-conserved cbm and the presence of a predicted chloroplast transit peptide suggested to us the possibility that the protein product of gene at5g participates in carbohydrate metabolism. we investigated this hypothesis further by examining patterns of correlated gene co-expression data as detailed in the methods section. we examined a set of genes known to be involved in starch metabolism given in ). we found that eight of these have correlation coefficients greater than r =; at5g; at3g; at1g; at4g; at1g; at1g; at1g ). we next examined the patterns of expression for at5g under various experimental conditions at the genevestigator site. it appeared that expression levels varied in a diurnal pattern. we have downloaded the original microarray data for the diurnal experiment of smith et al., and have compared the temporal expression patterns of at5g with the set of eight starch metabolic enzyme genes cited above whose general co-expression pattern are most highly correlated as revealed at the arabidopsis co-expression tool site. the results are shown in figure it is readily apparent that at5g and these starch metabolic enzymes show a similar overall diurnal pattern, with expression declining during the first half of the experiment, and rising to a maximum near the end of the experiment. the correlation coefficients between the expression pattern of at5g and each of these genes are as follows: at1g; at2g; at1g; at3g; at1g; at1g; at5g;at4g. the mean correlation coefficient for this set of data is r =. the presence of a strongly predicted coiled-coil domain in the amino acid sequence of at5g suggested to us the possibility of protein-protein interactions. we therefore screened the reference set of starch metabolic proteins for predictions of chloroplast transit peptides and cc domains. of the sequences proved to be negative for cc domains by all three tests. the other had various cc regions predicted, at different strengths, in the different methods. the evidence for the subset of proteins with the strongest cc predictions is presented in additional table and the full set of predictions is summarized in additional table there is at least moderate support by one prediction method, and reinforcement by a second method, for cc domains in five starch synthetic enzymes, at1g, at3g, at1g, at4g ) and two starch degradative enzymes, at1g ). the evidence is most definitive for sts and sts where strong predictions are obtained by all three methods for at least one region each. sts is especially notable in that the predicted coiled-coil region is exceptionally long. of the starch metabolic proteins set, failed to achieve a consensus positive chloroplast transit peptide prediction. all of these failures have been confirmed to be plastidic by experiment. in order to provide context for the general gene co-expression results with at5g we used the arabidopsis coexpression data mining tools site to obtain the correlation coefficients for all-against-all comparisons of the genes in the starch metabolic proteins set, over the full nasc microarray database. we set an arbitrary cutoff of r = and above to define the strongest class of gene co-expression correlations. we found, as one would expect, a pattern of highly correlated co-expression between genes encoding starch synthetic enzymes, and also between genes encoding starch degradative enzymes. however, we also observed a number of instances of unexpected high correlation between starch synthetic and starch degradative enzymes. the correlated gene groupings are depicted in additional table the numerical values for all the gene co-expression correlation comparisons are presented in additional table the most remarkable standout in this collection of data is at4g. it has strongly correlated co-expression with the largest number of other starch metabolic enzyme genes, and strong correlations with the largest number of starch degradative enzyme genes. carbohydrate binding preference of at5g39790 in order to test that at5g is a carbohydrate binding protein, as predicted by, a carbohydrate binding experiment was performed with at5g cloned, expressed and purified without the putative chloroplast transit peptide. the protein was incubated with starch, amylopectin or amylose, and was observed in the pellet fractions with all three carbohydrates by western blot analysis, as shown in figure these results confirm the prediction that this gene product is indeed a carbohydrate binding protein. the predicted molecular weight is close to, as expected due to the presence of the prset purification tag. although the protein bound all three carbohydrates, some unbound protein was observed in the supernatant of the amylopectin sample, indicating that at5g has a preference for the longer α 1→ linked glucose units found in amylose rather than the shorter regions found in α 1→ branched amylopectin. the binding of at5g to starch was also studied as a function ph, redox state and magnesium ion concentration. during the day the stromal ph is more alkaline, and redox conditions more reducing, than at night. as well, stromal magnesium ion concentrations fluctuate between mm during the day and mm at night. the results of this study, presented in figure show that at5g does not dissociate from starch as a function of ph, mg2+ concentration, or redox state. diurnal protein accumulation profile of at5g is correlated with microarray mrna transcript levels the low natural abundance of at5g in a. thaliana leaves made detection of signals on western blots difficult, so the spinach results are presented here. our ability to obtain a stronger signal in spinach is unclear and may simply reflect higher expression in spinach for some unknown reason. results in figure show that protein levels increase during the day and decrease at night over the course of hours when equal loading of fresh weigh leaf extracts were analyzed. focusing on the data between time points 12– h shows a direct correlation between the protein level and the a. thaliana mrna levels determined by microarray analysis. both fluctuate synchronously during day/night conditions. in a control western blot experiment using only the secondary antibody, no protein was detected. earlier research to monitor diurnal changes in starch metabolizing enzymes was also performed using fresh weight leaf extracts for both mrna analysis by microarray and transcript analysis by western blotting. these researchers showed that many proteins involved in starch biosynthesis had protein levels that stayed constant even when their mrna levels fluctuated diurnally. it was proposed that protein levels were under post-transcriptional control. an exception was granule-bound starch synthase, gbs a protein embedded in the starch matrix responsible for the synthesis of the amylose fraction of starch. protein levels for gbs increased during the day, and decreased at night as the starch granule was degraded. our results with at5g show a similar pattern, and this is not unexpected given the fact that dissociation from starch as a function of ph, mg2+ concentration, or redox state was not observed. chloroplast localization of at5g39790 the data presented in additional table of this study predicts that at5g is chloroplast localized. to test this prediction we performed three experiments. in the first experiment chloroplasts were isolated from spinach instead of a. thaliana and probed with antibodies for the presence of at5g results in figure show that our protein is indeed localized in the chloroplast fraction of spinach isolates and absent in the cytosolic fraction. the second experiment utilized a c-terminal gfp fusion construct of at5g that contained the putative chloroplast transit peptide. plasmids containing the gfp fusion construct or the plastid targeted red fluorescent protein control, were bombarded into onion epidermal cells. figure shows that after hours the gfp-at5g and rfp co-localize to the chloroplast. thus the presence of the putative transit peptide indeed targets at5g to this organelle. the third approach was to generate a transgenic arabidopsis thaliana plant expressing at5g39790-gfp. this shows a clear localization of the enzyme to chloroplasts as evidenced by chlorophyll fluorescence. discussion we have shown by experiment that the protein encoded by arabidopsis gene at5g is chloroplast-localized, and that it binds starch. this is consistent with bioinformatic analysis of the amino acid sequence, which predicts a chloroplast transit peptide, and a well-conserved c-terminal carbohydrate-binding domain. other structural analysis strongly predicts the presence of a coiled-coil domain. such regions most often consist of a repeating heptad pattern of regularly spaced, alternating hydrophobic and hydrophilic residues, resulting in an alpha-helical supercoil between partner protein interaction surfaces. many examples have been characterized, including motor and other cytoskeletal proteins, chromosomal and membrane organizing proteins, and transcription factors. the presence of both a starch-binding domain and a coiled-coil domain in the structure of at5g suggests the hypothesis that this is a scaffolding protein, whose structural properties would allow it to bind to the starch granule while also interacting with other proteins by way of its coiled-coil domain. analysis of gene expression patterns strongly supports the involvement of at5g in a coordinately-regulated set of carbohydrate-metabolizing proteins. the correlation coefficients are substantial for general co-expression across a large number of microarray experiments between at5g and several starch metabolic proteins. in particular, there is a clear diurnal pattern of expression which at5g shares with a set of starch metabolic proteins. smith et al in their original presentation of this diurnal experiment microarray data commented in particular on the marked co-expression pattern of a group of starch degradative enzyme-encoding genes. we find that at5g shares an evident pattern of co-expression with several of these degradative enzyme genes, but also with the gene for the synthetic enzyme sts. the role of sts has not been defined clearly, but is expected to function in starch synthesis. our structural analysis of a set of starch metabolic enzymes showed appreciable coiled-coil potential in several of their amino acid sequences. this was especially notable in the starch synthases. there is a body of previous work, both genetic and biochemical, showing the importance of multi-protein complexes in starch synthesis. recently, in wheat amyloplasts, homo-dimeric complexes between sbe isoforms have been observed, and hetero-multimeric complexes between ssi or ssii and sbe isoforms. in zea mays amyloplasts, a number of pair-wise interactions were demonstrated between starch synthetic enzymes. of particular interest are large complexes involving ssii and ssiii. the latter was shown to interact with ssi in a region that shares similarity with a number of other ssiiis from various plant species. this region aligns with the portion of the arabidopsis sts sequence containing cc regions, and in fact we have found that the zea mays sequence also contains cc potential in this region similar to the arabidopsis protein. hence cc interactions should be considered likely candidates to explain at least some of the multiple protein-protein interactions known to be occurring in the starch synthetic enzyme system. in our structural analysis, arabidopsis sts was distinctive in having the largest predicted cc region, probably extending more than residues. this indicates a high potential to participate in multiple cc interactions with other proteins. this might indicate that sts participates in a large multi-protein assembly. relatively little is known about the details of sts function. however, sts mutant plants were recently analyzed and it appears that this enzyme controls the number of starch granules in plastids, perhaps by initiating starch granule priming. the authors suggest that this is likely to occur in a large protein complex. in the present study we observed a surprising number of instances of correlated gene co-expression between starch synthetic and starch degradative enzyme genes. this was most marked for at4g, which has high correlations with a number of starch degradative enzyme genes. correlations of this type might be expected if starch synthetic enzymes and starch degradative enzymes were working together in a multi-protein complex. it has recently been shown that a heteromeric complex of stisa and stisa isoforms is necessary for the initiation of starch granule synthesis in potato. these authors concluded that it is likely that such a situation also occurs in arabidopsis. indeed, our gene expression data show a high correlation between the general expression patterns of at4g and at1g, with only a slightly weaker correlation with at2g. the presence of a number of other such correlations in our dataset might indicate that there are further, as yet undetected, interactions between starch synthetic and starch degradative enzymes. arabidopsis sts would appear to be a promising target for further investigations in this area. the combination of gene co-expression data, protein sequence structural analysis, and the results of the carbohydrate binding studies presented in this study strongly support the hypothesis that at5g is a scaffold protein closely involved in starch metabolism. the high degree of conservation of the carbohydrate-binding domain and coiled-coil regions of the sequences of the homologues collected from other plant species makes it very likely that they share the same function. however, sequence heterogeneity in the amino-terminal region of this protein set is observed, and chloroplast transit peptide predictions have provided mixed results. bioinformatic prediction of chloroplast transit peptides is a difficult process, since there is little conservation observed at the primary or secondary sequence level in actual transit peptides. indeed, in this study, of starch metabolic protein sequences in arabidopsis failed to attain the recommended performance of three out of four positive test predictions. all of these sequences except one are known to be plastid-localized. on a larger scale, the zybailov study identified proteins as plastid-localized with a high degree of confidence, but of these only 86% could be assigned chloroplast transit peptides by the targetp technique. the plastid localization of the at5g homologues, which currently cannot be assigned chloroplast transit peptides with confidence, will require experimental investigation. our western blot and fluorescent imaging results agree with the bioinformatics predictions that at5g localizes to the chloroplast. the predicted carbohydrate-binding domain of at5g has been confirmed with the starch binding studies. our data suggest that at5g preferentially docks to amylose. given the nature of starch it is unclear why we did not see complete binding to amylopectin given that starch is ~25% amylose. we can propose that there is simply adequate binding sites for the protein if 25% of the starch is amylose. it is possible that the branching of amylopectin actually hinders at5g binding and reflects a unique feature of at5g and this may play a role in defining its in vivo function. of interest is that this protein does not dissociate from starch as a function of ph, mg2+ concentration, or redox state. as such it is highly probable that this protein binds to starch under both day and night conditions, and functions as a scaffolding protein for other proteins to bind to via the cc domain. not all starch binding proteins bind starch tightly all the time. the binding of the dual-specificity protein phosphatase sex has been shown, in vitro to be regulated both by ph and redox potential. sex is found to bind starch during the day but not at night. in contrast, α-glucan water dikinase binds at night but not during the day. even more interesting is the observation that granule-bound starch synthase, a protein embedded in the starch matrix has protein levels which fluctuate diurnally in the same manner as at5g gbs does not have a carbohydrate-binding domain, and based on similar diurnal protein levels and starch localization, it is tempting to speculate that gbs1may be anchored by at5g future studies utilizing biochemical and molecular genetics approaches will address this question.  we have found that the cbm protein at5g is expressed in a diurnal fashion at both the transcript and protein level. at5g is conserved among plant species and outside of the cbm, the protein maintains a conserved coiled-coil domain that may act a as scaffold to bind other proteins. gene expression analysis shows a pattern that correlates well with the expression other starch metabolic enzymes. the expressed protein readily docks starch and analysis with an antibody generated against at5g shows the protein is localized to chloroplasts as is a gfp construct of the protein. 
similar analysis of the set of starch metabolic enzymes shows that several have strong to moderate coiled-coil potential. gene expression analysis shows strongly correlated patterns of co-expression between at5g and several starch metabolic enzymes. bioinformatic analysis demonstrates a diurnal pattern of gene expression, with an accumulation of transcript during the light cycle and decline during the dark cycle. a corresponding diurnal pattern of change in protein levels in leaves is also observed. sequence analysis shows that at5g has a strongly-predicted coiled-coil domain. we propose that at5g is a regulatory scaffold protein, persistently binding the starch granule, where it is positioned to interact by its coiled-coil domain with several potential starch metabolic enzyme binding-partners. we here show that at5g is chloroplast-localized, and binds starch, with a preference for amylose.
 starch accumulation and degradation in chloroplasts is accomplished by a suite of over enzymes. recent work has emphasized the importance of multi-protein complexes amongst the metabolic enzymes, and the action of associated non-enzymatic regulatory proteins. arabidopsis at5g encodes a protein of unknown function whose sequence was previously demonstrated to contain a putative carbohydrate-binding domain.  we here show that at5g is chloroplast-localized, and binds starch, with a preference for amylose. the protein persists in starch binding under conditions of ph, redox and mg+ concentrations characteristic of both the day and night chloroplast cycles. bioinformatic analysis demonstrates a diurnal pattern of gene expression, with an accumulation of transcript during the light cycle and decline during the dark cycle. a corresponding diurnal pattern of change in protein levels in leaves is also observed. sequence analysis shows that at5g has a strongly-predicted coiled-coil domain. similar analysis of the set of starch metabolic enzymes shows that several have strong to moderate coiled-coil potential. gene expression analysis shows strongly correlated patterns of co-expression between at5g and several starch metabolic enzymes.  we propose that at5g is a regulatory scaffold protein, persistently binding the starch granule, where it is positioned to interact by its coiled-coil domain with several potential starch metabolic enzyme binding-partners. 