BACKGROUND
potato  is one of the world’s most important crop plants.
potato tubers are rich in starch and contain minerals and vitamins as well as essential amino acids.
due to its nutrient composition and the high starch content, it serves as staple food, animal feed and feedstock for many industrial purposes including bioethanol production and food thickener.
moreover, tuber starch is used in the paper and textile industry.
potato starch is composed of two polymers, branched amylopectin and linear amylose.
starch synthesis occurs in plastids, where both polymers form semi-crystalline, water-insoluble granules.
it is generally accepted that starch is mainly synthesized from adp-glucose through the orchestrated action of several plastid-localized enzymes  <cit> .
an overview of starch metabolism in leaves and tubers is depicted in fig.
<dig> showing that there are many parallels between both tissues regarding the enzyme activities involved.
although the same overall enzymatic reactions have to be catalyzed in both, leaves and tubers, there are profound differences between both tissues.
in leaves, starch is synthesized and degraded diurnally, serving as a nocturnal energy resource to maintain energy supply for biological processes.
in potato tubers, starch accumulates during development and is stored over a long period of time.
it maintains the energy demand of the dormant tuber and fuels the outgrowth of new shoots after dormancy is broken.
in leaves, atp needed for starch biosynthesis is generated during photosynthesis.
in contrast, atp has to be imported into the plastids in tubers.
moreover, the origin of glucosyl donors for starch biosynthesis differs between phototrophic and heterotrophic tissues.
in leaf chloroplasts, the generation of adp-glucose is directly linked to the generation of photoassimilates within the calvin-benson-cycle  <cit> .
the glucosyl donor for starch biosynthesis in sink tubers is derived from sucrose which is transported via the phloem from the photosynthetically active leaf tissues to the developing tuber.
in the tuber, sucrose reaching the cytosol has to be converted to glucose 6-phosphate  which is subsequently imported into the amyloplast where it is further metabolized to adp-glc and starch.
these differences give reasons to assume that different regulatory mechanisms operate in leaves and tubers.fig.
<dig> proposed pathway of starch metabolism in leaves and tubers of potato.
a in source leaves, photoassimilates are generated in the calvin-benson-cycle.
in form of f6p, these can be converted to g1p in two subsequent steps catalyzed by pgi and pgm.
g1p serves as substrate for agpase for starch biosynthesis.
tps from the calvin-benson-cycle can also be transported to the cytosol via the tpt in exchange for pi where they can be metabolized e.g.
to sucrose.
b in the tuber, sucrose is cleaved by susy into udp-glucose and fructose.
udp-glucose is converted to g1p by ugpase.
g1p subsequently is transferred to g6p by cytosolic pgm which can be imported into the amyloplast by gpt.
in the amyloplast g6p is then reconverted into g1p by plastidial pgm and as such can serve as a substrate for starch biosynthesis.
for detailed reviews on starch metabolism see  <cit>  and references therein.
f6p, fructose-6-phosphate; tp, triose-phosphate; tpt, triose-phosphate/phosphate translocator; pi, inorganic phosphate; pgi, phosphoglucoisomerase; g6p, glucose 6-phosphate; g1p, glucose 1-phosphate; pgm, phosphoglucomutase; susy, sucrose synthase; ugpase, udp-glucose pyrophosphorylase; ppi, inorganic pyrophosphate; agpase, adp-glucose pyrophosphorylase; ppase, inorganic pyrophosphatase; ss, starch synthase; gbss, granule-bound starch synthase; sbe, starch branching enzyme; gwd, glucan, water dikinase; pwd, phosphoglucan, water dikinase; bam, beta-amylase; amy, alpha-amylase; sex <dig>  starch excess 4; lsf, like starch-excess four; dpe, disproportionating enzyme; pho, alpha-glucan phosphorylase; gpt, glucose 6-phosphate/phosphate translocator, ntt, nucleotide translocator; glt, glucose transporter; vgt, vacuolar glucose transporter; mex, maltose transporter; fk, fructokinase
many genes coding for enzymes involved in starch metabolism are organized in gene families.
members of these families may play distinct roles in starch biosynthesis and breakdown in source and sink tissues, respectively  <cit> .
in rice, this has been shown for isoforms of adp-glucose pyrophosphorylase , starch branching enzyme , starch phosphorylase , disproportionating enzyme , starch synthase  and debranching enzyme  by qrt-pcr analysis of leaf and endosperm tissues  <cit> .
regulatory mechanisms influencing activities of starch metabolic enzymes act at the post-translational level like protein-protein interactions, protein phosphorylation and redox regulation .
in addition, accumulation of starch metabolic enzymes is controlled at the transcriptional level.
transcript abundance of many starch genes is regulated by the circadian clock and by sugar availability  <cit> .
in arabidopsis leaves, expression of the gbss <dig> gene is controlled by two clock transcription factors , namely the myb-related circadian clock associated  <dig>  and late elongated hypocotyl   <cit> , while in rice endosperm, gbss was reported to be regulated by two interacting proteins belonging to the myc and erebp families  <cit> .
further evidence for transcriptional regulation of starch metabolism comes from barley, where a sugar-inducible tf, susiba <dig>  belonging to the wrky class of tfs, bound to the promoter of the isa <dig> gene and exhibited an expression pattern similar to isa <dig>  <cit> .
in a co-expression analysis in rice, putative regulators of starch biosynthesis were identified and subsequent functional studies showed that an apetala <dig> /erebp-type tf negatively regulates genes involved in starch biosynthesis and is an important determinant of the starch content and structure  <cit> .
in sweet potato, srf <dig>  a dof protein, was found to have an indirect positive effect on starch biosynthesis  <cit> .
an effect on starch gene expression was also described for flo <dig>  in rice seeds  <cit> .
these examples emphasize the significance of transcriptional regulation for starch metabolism which remains largely elusive in potato.
in potato, expression of sucrose synthase  and agpase can be induced in response to sucrose  <cit> .
moreover, the expression of susy and agpase are high in growing tubers and decrease rapidly after detachment of potato tubers from the mother plant  <cit> .
expression of both genes follows the diurnal rhythm in leaves and tubers  <cit> .
these findings suggest a coordinated regulation of both transcripts  <cit> .
analyses of overexpression-lines and antisense-lines point to an involvement of sucrose non-fermenting-1-related protein kinase- <dig>  in the regulation of transcription of susy and agpase  <cit>  but the molecular details are still unclear.
despite the important role of potato tubers for food security, a detailed study of the genomic organization of starch metabolic genes is still missing.
based on the recently published potato genome sequence  <cit> , genes encoding enzymes involved in starch metabolism in potato were identified and annotated in this study.
thereby, so far undescribed potential starch genes were identified and a functional genomic map of the starch gene inventory of the potato was established.
a comparative analysis of microarray and ngs data led to the identification of tissue-specifically expressed starch genes suggesting distinct regulatory mechanisms for leaf and tuber starch metabolism.
moreover, a co-expression analysis with tuber-specific starch genes was performed to identify transcription factors that may control starch biosynthesis in this tissue.
RESULTS
annotation of genes encoding enzymes of potato starch metabolism
in order to identify potato starch metabolism genes, a homology search using arabidopsis sequences of genes previously described by sonnewald and kossmann  <cit>  was conducted.
based on sequence similarity  <dig> out of  <dig> arabidopsis  open reading frames  were assigned to homologous potato transcripts .
no homologous sequences were found for at4g <dig> , at2g <dig>  and at5g <dig>  in the spud db  <cit>  or the ncbi databases.
for all other arabidopsis query sequences, a homologous sequence was found in the potato genome .table  <dig> starch metabolism genes investigated in this study
pgsc0003dmg400003495
pgsc0003dmt400008970
sotub11g <dig> .1
sotub11g <dig> .1
for the identification of isoforms of starch metabolic enzymes, a keyword search in the spud db database was undertaken using the enzyme names as queries.
additionally, manually corrected potato transcript sequences resulting from the homology and keyword searches were re-blasted against the potato genome and the sequences of second best hit were analyzed to identify putative isoforms.
this led to the discovery of two genes which had not been annotated, namely pgm <dig>  and pho2a.
pgm <dig>  could be assigned to chromosome  <dig> while pho2a was located on an unanchored scaffold.
eventually, predicted transcript sequences of all identified genes were compared to published mrna sequences available on the ncbi data base via a blast search.
sequence alignments were conducted to check for completeness of the orfs and the predicted protein sequences.
the exon-intron structure of the genes was manually re-annotated and/or corrected, if required.
correct assignment of potato transcripts compared to the corresponding arabidopsis orthologs was verified by protein sequence comparison.
phylogenetic trees were constructed using the translated orf sequences of all putative members of a gene family.
phylogenetic trees of selected gene families are depicted in additional file  <dig>  if ambiguities were encountered, a motif search was conducted using the online tool meme  <cit> .
the presence and order of motifs was compared between sequences assuming a high degree of similarity between members of the same gene family  <cit> .
if this was the case the identified gene was considered as an isoform.
application of the above mentioned strategies resulted in the identification of  <dig> loci coding for enzymes of starch metabolism in potato .
in comparison to arabidopsis, additional putative isoforms of agps <dig>  pho <dig> and pho <dig>  tpt, bam <dig>  bam <dig>  sbe <dig>  gpt <dig> and gpt <dig>  ppase, isa <dig>  sex <dig>  pgm <dig>  pgi, amy <dig> and amy <dig> were found.
the deduced transcripts of bam <dig> , bam <dig> , sbe <dig>  and isa <dig>  were highly identical to their respective paralogs but did not seem to comprise full-length transcripts.
this might be either a result of an incorrect genome assembly or incomplete gene duplication events.
chromosomal positions of putative starch genes were retrieved from the spud db genome browser v <dig>   <cit>  and visualized using the location-based display tool on the ensembl plants website  <cit> .
manual editing allowed the visualization of genes as an ideogram .
for two genes, pho1a and pho2a, no physical position could be defined since their genes are located on unanchored scaffolds, but orthologous sequences from tomato are located on chromosomes  <dig> and  <dig>  respectively.
this is in accordance with results from quantitative trait loci  analyses in potato that mapped two glucan-phosphorylases to those chromosomes  <cit> .fig.
<dig> ideogram of physical positions of starch metabolism enzymes in the potato genome.
the relative map positions of  <dig> genes encoding starch metabolism genes are shown on the individual pseudomolecules depicting the chromosomes 1–12
figure  <dig> shows that genes coding for starch metabolism enzymes are distributed over all twelve potato chromosomes.
there is a concentration of ss  on chromosome  <dig> and many genes encoding bams are located on chromosome  <dig>  susy and isa encoding genes are distributed across different chromosomes.
an interesting finding was the discovery of two pgm <dig> isoforms pgm <dig>  and pgm <dig>  which are located on chromosome  <dig> and  <dig>  respectively.
the sequence identity between both transcripts is  <dig> %, but the corresponding genes differ significantly in their non-coding regions showing only 59% sequence similarity.
however, the structure of both genes appears to be conserved.
the pgm <dig>  isoform has not been predicted by the pgsc or itag and was identified by blasting the transcript sequence sotub07g <dig> .
<dig> against the scaffold sequences.
investigating the tomato genome available on the sol genomics website  for pgm <dig> genes revealed that only one locus is present which is localized on chromosome  <dig>  therefore, it is conceivable that the pgm <dig>  gene on chromosome  <dig> is the result of a recent gene duplication event, however this needs to be further investigated by bioinformatics analysis.
identification of suitable microarray identifiers to investigate gene expression
two oligonucleotide-based microarray platforms  are available for global gene expression analysis in potato.
the poci array was designed in 4x44k format based on a collection of expressed sequence tags   <cit>  while the 8x60k microarray is based on predicted transcript sequences of the dm potato genome by the pgsc  <cit> .
in this study, experimental data of both microarray designs were used.
the prerequisite for the comparative expression analysis was the identification of suitable microarray oligonucleotides  matching the transcript of interest, particularly in case of the poci platform.
therefore, prior to expression analysis, oligonucleotide binding accuracy to the target genes was assessed.
to this end, transcript and genomic sequences of starch genes were blasted against the poci database  <cit>  and resulting est sequences were aligned to the genomic sequence to allow for assessment of their corresponding oligonucleotide binding capacities to the transcript.
oligonucleotides matching the reference sequence with 85% or more identity were considered for the analysis of expression profiles.
due to the lack of matching est-sequences or to binding of the corresponding oligonucleotides within predicted introns, no suitable oligonucleotides were found for amy3-like, agps <dig> , agps <dig>  tpt, tpt-like, gpt <dig> , all bam <dig> isoforms, bam <dig>  bam <dig>  pgi-like <dig> and ppgm in the poci platform.
since oligonucleotide sequences of the 8x60k microarray were deduced from predicted transcript sequences of the dm genome, they perfectly match the corresponding transcript available at the spud db website.
in these cases the position of the oligo within the gene was assessed to rule out that the binding site is within a putative intron.
oligonucleotide specificity was investigated by multiple sequence alignments.
the high sequence similarity between the transcripts of some isoenzymes prevented the assignment of specific oligonucleotides discriminating the isoforms of isa <dig>  sex4-like, sbe <dig> and bam <dig>  and bam <dig> .
additional file  <dig> lists all identifiers from both platforms that met our criteria and that were considered for further analyses.
identification of genes that are highly expressed in leaves or tubers
for the gene expression analysis, samples taken from leaf and tuber tissues were selected from different microarray experiments .
raw data files of the different samples were uploaded into the genespring  <dig> .
<dig>  gx software and were normalized together.
direct comparisons of gene expression were made within the individual platforms first.
afterwards derived results were compared between the different platforms.
to identify starch genes that are preferentially expressed in leaves or tubers, the fold-change between the mean relative expression value detected in leaf and tuber samples was calculated using the genespring  <dig> .
<dig>  gx software and displayed in additional file  <dig>  for genes, whose expression was ascertainable in both microarray platforms, the log <dig> fold-change was calculated and depicted in fig.
<dig>  we considered genes that were on average more than 10-fold overexpressed in one tissue to be tissue-specific.
the comparison between the two array platforms revealed that several genes are specifically expressed in leaves or tubers, respectively .
hence, a strong tuber-specific expression was detected for gpt <dig>  and susy <dig> followed by sex <dig> and ss <dig>  whereas bam <dig> , apl <dig> and amy <dig>  were found to be highly expressed in leaves.
fold-change differences between leaf and tuber samples were often greater in the 8x60k array than in the poci array but the tendency was similar .
the only exception was gpt <dig>  whose expression was unchanged between leaf and tuber samples hybridized onto the poci array but showed a  <dig> -fold higher expression in leaves than in tubers in samples analyzed on the 8x60k array .fig.
<dig> relative expression of starch genes in leaf vs. tuber tissue.
fold-change values for individual genes between leaf and tuber samples were exported from genespring or calculated from fpkm values from the pgsc database.
genes whose expression cannot be detected in either platform were excluded.
light grey bars: values from 8x <dig> microarray, black bars: values from 4x44k poci array, dark grey bars: fpkm values
to confirm our results, fpkm  values of corresponding genes were downloaded from rna-sequencing data available on the spud db website and leaf and tuber samples were selected.
ratios between leaf and tuber values were calculated and compared to the results from the microarray analyses.
fold-change values of the rna-seq data compared well to the microarray data .
thus, gpt <dig>  and susy <dig> are highly tuber-specifically expressed genes.
their expression was 20- to 1000-fold higher in tubers compared to leaves.
leaf-specific expression of amy <dig> , apl <dig> and bam <dig>  could also be confirmed by the rna-seq data.
they were found to be 7-fold to 320-fold higher expressed in leaves than in tubers .
verification of differential expression of selected genes was carried out by quantitative real-time pcr .
as shown in fig.
<dig>  tuber-specific expression was confirmed for susy <dig>  gpt <dig>  and ss <dig> as well as the leaf-specific expression of amy <dig> , apl <dig> and bam <dig>  .
in addition, we selected two genes, apl <dig> and lsf <dig>  showing a similar expression in leaves and tubers in all three transcriptome platforms.
again, qrt-pcr analysis confirmed the transcriptome data .fig.
<dig> qrt-pcr analysis of selected starch metabolism genes in potato leaves and tubers.
plants were grown in a greenhouse for 11 weeks until harvest and sampling.
mean relative expression of four biological replicates normalized to ef1alpha is illustrated as dct-value of a susy <dig>  b apl <dig>  c gpt <dig> , d bam <dig> , e ss <dig>  f amy <dig> , g lsf <dig>  h apl <dig>  error bars represent standard deviation
selection of query genes for co-expression analysis
the main goal of the co-expression analysis  was to identify possible regulators of starch biosynthesis in potato tubers.
therefore, the genes used as queries for the analysis were selected by two criteria; first, they had to be specifically expressed in the tuber and second, their expression pattern had to follow starch accumulation.
the first criterion was fulfilled most strongly by gpt <dig> , susy <dig>  sex <dig>  ss <dig> and sbe <dig> .
for the evaluation of the second criterion, increasing gene expression during tuber development was chosen.
it is known that during tuberization the rate of starch biosynthesis increases significantly  <cit> .
therefore, genes involved in starch biosynthesis should be upregulated during this process.
to identify these genes, microarray data from the tuber induction experiment described by ferreira et al.
<cit>  were inspected and the ratio of transcripts detected in small tubers  vs. those measured in unswollen stolons  were calculated and illustrated as log <dig> values .
the highest up-regulation from stage  <dig> to stage  <dig> was seen for susy <dig>  sbe <dig>  gpt <dig>  and lde.
sex <dig>  which was identified as specifically expressed in tubers, showed a pronounced down-regulation in the course of tuber development .
therefore, susy <dig>  sbe <dig> and gpt <dig>  were chosen as query genes for the co-expression analysis.fig.
<dig> relative changes in expression of starch genes during tuber development .
given are log <dig> transformed fold-changes.
data were taken from ferreira et al.
<dig>  <cit>
co-regulation analysis to identify putative regulators of starch metabolism in potato tubers
to identify possible regulators of starch biosynthesis in potato tubers, all valid microarray identifiers for each of the selected genes  were used as queries in a pearson correlation search on all detected entities in both microarray platforms including all data sets.
in addition, rna-seq data were also analyzed.
a pearson correlation coefficient  of  <dig>  was used as cut-off .
within each platform, the overlap of entities co-expressed with all three query genes was determined using venn diagrams .
the numbers of genes co-regulated with gpt <dig> , susy <dig> and sbe <dig> differed greatly between platforms ranging between  <dig> entities in the poci array,  <dig> for the rna-seq data set and  <dig> in the 8x60k array .
to compare the results from the different platforms, found entities were assigned to their corresponding pgsc gene identification number.
this resulted in a list of  <dig> different genes that were consistently co-expressed with gpt <dig> , susy <dig> and sbe <dig>  besides the three query genes, five other starch genes, namely apl <dig>  pho1a, ss <dig>  ntt <dig> and gpt <dig>  were among the co-expressed genes  and were identified to be tuber-specifically expressed .
functional categorization of the  <dig> co-expressed genes revealed that twenty percent of the co-expressed genes encode known storage proteins like patatin and protease inhibitors  <cit> .fig.
<dig> overview of co-expression analysis.
a-c venn-analysis of co-expressed entities with susy <dig> , sbe <dig>  and gpt <dig>  .
co-expression analysis was conducted using a pcc cut-off of  <dig>  < =r < = <dig> .
a co-expressed entities in the poci microarray platform, b co-expressed entities in the 8x60k microarray platform, c co-expressed entities in the rna-sequencing data.
d after conversion of the co-expressed entity lists to gene lists, the lists were compared and the common genes in all three lists were retrieved
to identify possible transcriptional regulators of starch biosynthesis in potato tubers, we paid special attention to putative tfs.
among those, tfs with homology to regulators of organogenesis from arabidopsis like petal loss , lateral organ boundaries , blade on petiole <dig>  and lateral root primordium protein  were found.
furthermore, a wrky-type tf  and a member of the plant-specific tify  motif containing protein family tify5a, were co-expressed with the starch biosynthesis genes .
to confirm the expression profiles, four putative tf  as well as susy <dig> and gpt <dig>  were selected for qrt-pcr analysis.
the relative amount of the corresponding mrna was quantified in an independent set of samples representing four different stages of tuber development, namely unswollen stolons , swollen stolons , growing tubers and dormant tubers.
the results were compared to microarray data derived from similar stages of tuber development .
as shown in fig.
<dig>  the results from qrt-pcr were generally comparable to the results from microarray analysis when considering similar stages of tuber development.
with respect to susy <dig> slight differences between both techniques were observed.
while its expression reached highest values in stage  <dig> in the microarray, a maximum transcript amount of susy <dig> was seen in growing tubers in qrt-pcr.
the expression profiles of lob followed those of susy <dig> in both setups.
wrky <dig> as well as tify5a showed similar profiles in both platforms and correlated highly to the expression of gpt <dig>  .
one exception was the expression profile of ptl.
while its expression was lower in growing and non-growing tubers as compared to stage  <dig> in the microarray experiments, the mrna level increased steadily across all developmental stages in the qrt-pcr reaching its maximum in dormant tubers .fig.
<dig> expression profiles of tuber-specific starch genes and co-expressed transcription factors.
a gene expression calculated as fold-change relative to the value at stage  <dig> in the microarray experiments.
b qrt-pcr analysis of the same genes in independent samples.
each value represents the mean of 3– <dig> biological replicates.
error bars represent standard deviation.
black bars: susy <dig>  dark grey bars: gpt <dig> , grey bars: ptl, medium grey bars: tify5a, light grey bars: lob domain containing protein, white bars: wrky4
a pearson correlation matrix was constructed evaluating the similarity of the expression profiles determined by qrt-pcr .
most pcc values were greater than  <dig>  indicating that selected genes were co-regulated in the samples analyzed by qrt-pcr.
however, the pcc values were lower than in the global co-expression analysis which is most likely due to the decreased sample number.
thus, the qrt-pcr analysis corroborated that expression profiles of tify5a, lob and wrky <dig> are similar to those of susy <dig> and gpt <dig>  during tuber development.
for ptl, the pccs calculated from qrt-pcr were low supporting the observation that the expression pattern of this gene in the samples used for qrt-pcr deviated from the microarray.table  <dig> pearson correlation coefficients between starch genes and tfs based on qrt-pcr analysis
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
results from the qrt-pcr analysis of starch genes and transcription factors were subjected to a pearson correlation analysis using microsoft excel.
correlation coefficients with p ≤  <dig>  are indicated in bold letters
discussion
genome-wide analysis of starch genes in potato reveals novel isoforms
enzymes involved in starch metabolism often belong to gene families encoding several isoenzymes.
to our knowledge, this work presents the first genome-wide analysis of starch genes in potato.
a comprehensive blast search strategy complemented by motif discovery and comparison to known sequences from arabidopsis was applied aiming at the identification of all “starch gene” loci in potato.
we found  <dig> loci coding for starch metabolism related enzymes belonging to different enzyme classes.
higher plants possess five gene classes encoding starch synthases, designated gbss and ss1- <dig>  <cit> .
in rice, two forms of gbss were identified and eight genes encoding the four ss classes  <cit> .
in the potato genome, we confirmed that gbss which has been reported previously to be responsible for amylose biosynthesis in the starch granule, is encoded by a single gene and is expressed higher in tubers than in leaves  <cit> .
a second transcript  annotated as gbss <dig> in the pgsc database had previously been described to possess soluble ss activity  <cit>  and was found to be the closest potato homolog to arabidopsis ss <dig> and was therefore consequently designated as ss <dig>  moreover, it was shown that ss <dig> plays only a minor role in starch biosynthesis in tubers  <cit>  which is in accordance with our expression analysis showing only a slightly higher expression in tubers compared to leaves although being upregulated during tuber development.
in total, seven starch synthases were found in the potato genome  most of which have been described in earlier studies .
however, no studies have analyzed the roles of ss <dig> and ss <dig> in potato yet, but our gene expression data suggest a possible role for ss <dig> in potato tuber starch biosynthesis.
in contrast, ss <dig> is expressed to similar levels in leaves and tubers  and its expression was not found to change significantly during tuber development .
thus, further analyses are necessary to investigate the function of these genes during starch biosynthesis in potato.
a recent publication described the phylogenetic relationship of ss from different plant species, including potato, confirming the presence of a fifth class of ss  <cit> .
in addition the authors found maize ss <dig> to be highly expressed during the grain filling stage suggesting a role in starch biosynthesis  <cit> , which is in agreement with our assumption.
in this study, enzymes were designated regarding to their annotation in arabidopsis.
in most cases, this was in accordance with isoform numeration of already described enzymes of potato.
one exception concerns the numeration of isoforms within the sbe class where we identified four isoenzymes.
two of them share a very high sequence similarity to each other and have been denoted as sbe <dig>  and sbe <dig>  due to their homology to arabidopsis sbe <dig>  the deduced transcript sequences of these two genes, however, do not correspond to the previously published potato sbe <dig> sequences  <cit> .
the gene product designated sbe <dig> in the aforementioned studies corresponds to sbe <dig> in this study.
it was described as the major sbe isoform in potato tubers and was found to play a role in starch granule formation  <cit> .
this is in accordance with the expression profile during tuber development and tissue preference discovered in this study.
until now, only variants of two isoforms, sbe <dig> and sbe <dig>  have been shown to act as branching enzymes in the amyloplast  <cit> .
the role of the two potato sbe <dig> paralogs identified in this study remains unclear.
in arabidopsis, sbe <dig> has an effect on embryogenesis and is essential for plant growth and development  <cit> .
a direct implication of atsbe <dig> in starch metabolism is unacquainted.
comparative microarray analysis revealed tissue-specific gene expression
to identify tuber- and leaf-specifically expressed starch genes different microarray data sets were analyzed.
to enable the analysis, specific microarray probes had to be assigned to the different starch genes and their respective isoforms.
in general, our findings were in agreement with previously published gene expression analyses and showed a high reproducibility between the two microarray platforms.
tissue-specific expression of enzyme isoforms was for example found for pho1a and pho1b.
pho1b appeared to be preferentially expressed in leaves, while pho1a was expressed higher in tubers, which is in accordance with previous findings  <cit> .
in the case of agpase, most subunits are expressed slightly higher in tubers than in leaves according to our results.
however, one isoform, namely apl <dig>  was clearly expressed higher in leaves than in tubers.
this is in contrast to findings from la cognata et al.
<cit>  who described tuber-specific expression of apl <dig> .
the reliability of our results was confirmed by rna-seq data and by qrt-pcr using leaf and tuber samples.
genes showing tuber-specific expression were susy <dig>  sbe <dig>  ss <dig>  gpt <dig>  and sex <dig>  in contrast to the other tuber-specific isoforms, sex4-specific transcripts were not up-regulated during tuber development which is consistent with the proposed role of the enzyme in starch degradation  <cit> .
the activity of the main susy isoform in tubers, susy <dig>  is connected to the onset of tuberization  and correlates well with transcript and tuber starch accumulation in potato  <cit> .
accordingly, susy <dig> overexpression led to an increased starch content and higher tuber yield in potato plants  <cit>  supporting its suggested key role in starch metabolism.
similarly, sbe <dig> and gpt <dig>  expression have been linked to tuber development and the accompanying accumulation of starch .
in this context, overexpression of gpt <dig>  together with ntt resulted in increased tuber starch content and yield  <cit>  indicating that expression and activity of gpt <dig>  are closely related.
the similarity between the expression patterns of these enzymes strongly supports the assumption of a coordinated transcriptional regulation of genes within the same pathway  <cit> .
moreover, these examples confirm that enzymatic activity of susy and gpt <dig>  nicely correlates with transcript accumulation and that accumulation of starch metabolic enzymes is controlled at the transcriptional level.
however, in other species activity of starch metabolic enzymes was shown to be additionally regulated by post-translational mechanisms.
for example, phosphorylation of susy isoforms was shown to influence sub-cellular localization and protein stability  <cit> .
activity of sbe isoforms was reported to be regulated by protein phosphorylation and redox state  <cit> .
co-expression analysis reveals candidate regulators of starch biosynthesis
co-expression analysis has previously been described to be a suitable tool for the identification of co-regulated genes  <cit> .
assuming that proteins with regulatory functions have to be expressed at the same time or shortly before their target genes, the identification of candidate regulators should be possible by co-expression analysis.
the great potential of this strategy has already been demonstrated in several studies including different plant species and tissues .
one example is the identification of rice starch regulator  <dig>  by fu and xue  <cit>  in a co-expression analysis similar to the approach used in this study.
rsr <dig> was found to be negatively co-expressed with rice starch synthesis genes and was experimentally verified as a modulator of starch gene expression.
in this work, genes that were identified as being tuber-specifically expressed and exhibiting an expression pattern that coincides with starch biosynthesis in the potato tuber were used to search for potential transcriptional regulators, since they are so far not known.
the number of genes identified to be co-expressed with susy <dig>  gpt <dig>  and sbe3differed between the two microarray platforms, and was about  <dig> times higher in the 8x60k experiments than in those performed with the poci platform.
one reason for this might be the sample selection of the 8x60k platform which basically consists of tuber samples in similar developmental stages while most samples taken from the 4x44k format were originally designed to reflect starch biosynthesis during tuber formation.
therefore, expression profiles derived from experiments using the poci array were expected to be more specific with respect to the identification of putative regulators of starch biosynthesis in potato tubers.
moreover, we reasoned that co-expression of a regulator with its target genes should occur in all situations.
thus, candidate selection was made after comparing the results of the co-expression analyses of the three query genes in three different platforms each with many individual samples.
eventually we identified  <dig> genes that are consistently co-regulated with susy <dig>  gpt <dig>  and sbe <dig>  inspection of co-expressed genes revealed a strong over-representation of genes involved in primary carbon metabolism and development as well as genes encoding storage proteins.
tuber development and storage metabolism are known to be highly associated processes  <cit>  which strengthens the significance of the retrieved candidates.
beside this, putative tfs co-expressed with the selected starch genes could be identified.
they belong to different classes and none of them has been characterized in potato so far.
clearly, there is a strong enrichment of tfs associated with developmental processes and organogenesis like bop <dig>  lob, ptl and lrp.
for ptl, a co-expression with susy <dig> and gpt <dig>  in samples representing different tuber developmental stages could not be confirmed via qrt-pcr and ptl might therefore not be a good candidate for further analysis.
the expression profiles obtained by qrt-pcr of the other three tf were in accordance with those of the microarray analysis .
slight variations between qrt-pcr and microarray were found when comparing expression levels of susy <dig> or lob on “stage 5” and “grow” from the microarray to “swollen stolon” and “growing tuber” samples used for qrt-pcr.
in the microarray, highest gene expression was seen on “stage 5”, while in the qrt-pcr expression peaked in growing tubers .
nevertheless, an increasing expression level was always associated with tuber formation.
a possible explanation for this disagreement might be slightly different developmental stages of the samples used for the analyses.
for the microarray defined stages of tuber development were sampled  <cit> , while for the qrt-pcr swollen stolons of different developmental stages were pooled.
furthermore, the growing tubers for the microarray experiment were monitored by x-ray ct analysis to determine their growth velocity, while the tubers sampled for qrt-pcr were taken from plants during their growth period, assuming that the tubers were still growing.
despite these small differences between different experiments, expression levels of lob, tify5a and wrky <dig> correlate well with susy <dig> and gpt <dig>  .
thus they might be interesting candidates for further analyses.
in arabidopsis, bop <dig> and its close homolog bop <dig> regulate the expression of lob-domain containing proteins  <cit> .
lob expression has been found in the boundary regions between meristematic tissue and developing lateral organ primordia of the shoot apical meristem and the roots  <cit> .
a similar spatial expression is exhibited by lrp <dig> of arabidopsis which has been shown to be expressed in root primordia in early developmental stages  <cit> .
in maize the localization of lrp in lateral root primordia was confirmed and it was demonstrated that lrp expression was auxin-inducible  <cit> .
a link to auxin-signaling may also be established by the closest homolog of potato wrky <dig> in arabidopsis.
based on sequence similarity, the closest homolog in arabidopsis is wrky <dig> which has been linked to auxin-signaling in root development  <cit> .
a role of auxin in tuber initiation has been suggested  <cit>  but a direct link to starch biosynthesis is missing.
the expression patterns of these tfs suggest that there are interesting candidate genes which may directly or indirectly control starch biosynthesis and that more detailed investigation of their role is worthwhile.
CONCLUSIONS
in this study the complete inventory of starch metabolism genes and their genomic localization was described which will facilitate future examinations of the distinct functions of isoenzymes in this pathway.
moreover, novel as far undescribed enzyme isoforms were identified whose characterization will shed more light on the mechanisms of starch biosynthesis and degradation in potato plants.
comparative microarray analysis uncovered leaf- and tuber-specific starch gene isoforms.
this finding suggests distinct regulatory mechanisms in transitory and storage starch metabolism.
a co-expression analysis was conducted using tuber-specific genes aiming at the identification of regulators of starch biosynthesis in potato tubers.
forty genes showed strong co-regulation in all platforms analyzed.
among the co-expressed genes were many storage metabolism genes belonging to the starch biosynthesis pathway or storage proteins as well as tfs.
none of the identified tfs had been described in potato yet, but many of their homologs in arabidopsis are known regulators of lateral organ development.
we conclude that tuber development and tuber starch biosynthesis are highly connected pathways and consider it worthwhile to investigate the influence of the identified regulators on starch biosynthesis.

 potato is one of the world’s most important crop plants. potato tubers are rich in starch and contain minerals and vitamins as well as essential amino acids. due to its nutrient composition and the high starch content, it serves as staple food, animal feed and feedstock for many industrial purposes including bioethanol production and food thickener. moreover, tuber starch is used in the paper and textile industry. potato starch is composed of two polymers, branched amylopectin and linear amylose. starch synthesis occurs in plastids, where both polymers form semi-crystalline, water-insoluble granules. it is generally accepted that starch is mainly synthesized from adp-glucose through the orchestrated action of several plastid-localized enzymes. an overview of starch metabolism in leaves and tubers is depicted in fig.  showing that there are many parallels between both tissues regarding the enzyme activities involved. although the same overall enzymatic reactions have to be catalyzed in both, leaves and tubers, there are profound differences between both tissues. in leaves, starch is synthesized and degraded diurnally, serving as a nocturnal energy resource to maintain energy supply for biological processes. in potato tubers, starch accumulates during development and is stored over a long period of time. it maintains the energy demand of the dormant tuber and fuels the outgrowth of new shoots after dormancy is broken. in leaves, atp needed for starch biosynthesis is generated during photosynthesis. in contrast, atp has to be imported into the plastids in tubers. moreover, the origin of glucosyl donors for starch biosynthesis differs between phototrophic and heterotrophic tissues. in leaf chloroplasts, the generation of adp-glucose is directly linked to the generation of photoassimilates within the calvin-benson-cycle. the glucosyl donor for starch biosynthesis in sink tubers is derived from sucrose which is transported via the phloem from the photosynthetically active leaf tissues to the developing tuber. in the tuber, sucrose reaching the cytosol has to be converted to glucose 6-phosphate which is subsequently imported into the amyloplast where it is further metabolized to adp-glc and starch. these differences give reasons to assume that different regulatory mechanisms operate in leaves and tubers.fig. proposed pathway of starch metabolism in leaves and tubers of potato. a in source leaves, photoassimilates are generated in the calvin-benson-cycle. in form of f6p, these can be converted to g1p in two subsequent steps catalyzed by pgi and pgm. g1p serves as substrate for agpase for starch biosynthesis. tps from the calvin-benson-cycle can also be transported to the cytosol via the tpt in exchange for pi where they can be metabolized e.g. to sucrose. b in the tuber, sucrose is cleaved by susy into udp-glucose and fructose. udp-glucose is converted to g1p by ugpase. g1p subsequently is transferred to g6p by cytosolic pgm which can be imported into the amyloplast by gpt. in the amyloplast g6p is then reconverted into g1p by plastidial pgm and as such can serve as a substrate for starch biosynthesis. for detailed reviews on starch metabolism see and references therein. f6p, fructose-6-phosphate; tp, triose-phosphate; tpt, triose-phosphate/phosphate translocator; pi, inorganic phosphate; pgi, phosphoglucoisomerase; g6p, glucose 6-phosphate; g1p, glucose 1-phosphate; pgm, phosphoglucomutase; susy, sucrose synthase; ugpase, udp-glucose pyrophosphorylase; ppi, inorganic pyrophosphate; agpase, adp-glucose pyrophosphorylase; ppase, inorganic pyrophosphatase; ss, starch synthase; gbss, granule-bound starch synthase; sbe, starch branching enzyme; gwd, glucan, water dikinase; pwd, phosphoglucan, water dikinase; bam, beta-amylase; amy, alpha-amylase; sex starch excess 4; lsf, like starch-excess four; dpe, disproportionating enzyme; pho, alpha-glucan phosphorylase; gpt, glucose 6-phosphate/phosphate translocator, ntt, nucleotide translocator; glt, glucose transporter; vgt, vacuolar glucose transporter; mex, maltose transporter; fk, fructokinase many genes coding for enzymes involved in starch metabolism are organized in gene families. members of these families may play distinct roles in starch biosynthesis and breakdown in source and sink tissues, respectively. in rice, this has been shown for isoforms of adp-glucose pyrophosphorylase, starch branching enzyme, starch phosphorylase, disproportionating enzyme, starch synthase and debranching enzyme by qrt-pcr analysis of leaf and endosperm tissues. regulatory mechanisms influencing activities of starch metabolic enzymes act at the post-translational level like protein-protein interactions, protein phosphorylation and redox regulation. in addition, accumulation of starch metabolic enzymes is controlled at the transcriptional level. transcript abundance of many starch genes is regulated by the circadian clock and by sugar availability. in arabidopsis leaves, expression of the gbss gene is controlled by two clock transcription factors, namely the myb-related circadian clock associated and late elongated hypocotyl, while in rice endosperm, gbss was reported to be regulated by two interacting proteins belonging to the myc and erebp families. further evidence for transcriptional regulation of starch metabolism comes from barley, where a sugar-inducible tf, susiba belonging to the wrky class of tfs, bound to the promoter of the isa gene and exhibited an expression pattern similar to isa. in a co-expression analysis in rice, putative regulators of starch biosynthesis were identified and subsequent functional studies showed that an apetala /erebp-type tf negatively regulates genes involved in starch biosynthesis and is an important determinant of the starch content and structure. in sweet potato, srf a dof protein, was found to have an indirect positive effect on starch biosynthesis. an effect on starch gene expression was also described for flo in rice seeds. these examples emphasize the significance of transcriptional regulation for starch metabolism which remains largely elusive in potato. in potato, expression of sucrose synthase and agpase can be induced in response to sucrose. moreover, the expression of susy and agpase are high in growing tubers and decrease rapidly after detachment of potato tubers from the mother plant. expression of both genes follows the diurnal rhythm in leaves and tubers. these findings suggest a coordinated regulation of both transcripts. analyses of overexpression-lines and antisense-lines point to an involvement of sucrose non-fermenting-1-related protein kinase- in the regulation of transcription of susy and agpase but the molecular details are still unclear. despite the important role of potato tubers for food security, a detailed study of the genomic organization of starch metabolic genes is still missing. based on the recently published potato genome sequence, genes encoding enzymes involved in starch metabolism in potato were identified and annotated in this study. thereby, so far undescribed potential starch genes were identified and a functional genomic map of the starch gene inventory of the potato was established. a comparative analysis of microarray and ngs data led to the identification of tissue-specifically expressed starch genes suggesting distinct regulatory mechanisms for leaf and tuber starch metabolism. moreover, a co-expression analysis with tuber-specific starch genes was performed to identify transcription factors that may control starch biosynthesis in this tissue.  annotation of genes encoding enzymes of potato starch metabolism in order to identify potato starch metabolism genes, a homology search using arabidopsis sequences of genes previously described by sonnewald and kossmann was conducted. based on sequence similarity out of arabidopsis open reading frames were assigned to homologous potato transcripts. no homologous sequences were found for at4g, at2g and at5g in the spud db or the ncbi databases. for all other arabidopsis query sequences, a homologous sequence was found in the potato genome.table starch metabolism genes investigated in this study pgsc0003dmg400003495 pgsc0003dmt400008970 sotub11g.1 sotub11g.1 for the identification of isoforms of starch metabolic enzymes, a keyword search in the spud db database was undertaken using the enzyme names as queries. additionally, manually corrected potato transcript sequences resulting from the homology and keyword searches were re-blasted against the potato genome and the sequences of second best hit were analyzed to identify putative isoforms. this led to the discovery of two genes which had not been annotated, namely pgm and pho2a. pgm could be assigned to chromosome while pho2a was located on an unanchored scaffold. eventually, predicted transcript sequences of all identified genes were compared to published mrna sequences available on the ncbi data base via a blast search. sequence alignments were conducted to check for completeness of the orfs and the predicted protein sequences. the exon-intron structure of the genes was manually re-annotated and/or corrected, if required. correct assignment of potato transcripts compared to the corresponding arabidopsis orthologs was verified by protein sequence comparison. phylogenetic trees were constructed using the translated orf sequences of all putative members of a gene family. phylogenetic trees of selected gene families are depicted in additional file if ambiguities were encountered, a motif search was conducted using the online tool meme. the presence and order of motifs was compared between sequences assuming a high degree of similarity between members of the same gene family. if this was the case the identified gene was considered as an isoform. application of the above mentioned strategies resulted in the identification of loci coding for enzymes of starch metabolism in potato. in comparison to arabidopsis, additional putative isoforms of agps pho and pho tpt, bam bam sbe gpt and gpt ppase, isa sex pgm pgi, amy and amy were found. the deduced transcripts of bam, bam, sbe and isa were highly identical to their respective paralogs but did not seem to comprise full-length transcripts. this might be either a result of an incorrect genome assembly or incomplete gene duplication events. chromosomal positions of putative starch genes were retrieved from the spud db genome browser v and visualized using the location-based display tool on the ensembl plants website. manual editing allowed the visualization of genes as an ideogram. for two genes, pho1a and pho2a, no physical position could be defined since their genes are located on unanchored scaffolds, but orthologous sequences from tomato are located on chromosomes and respectively. this is in accordance with results from quantitative trait loci analyses in potato that mapped two glucan-phosphorylases to those chromosomes.fig. ideogram of physical positions of starch metabolism enzymes in the potato genome. the relative map positions of genes encoding starch metabolism genes are shown on the individual pseudomolecules depicting the chromosomes 1–12 figure  shows that genes coding for starch metabolism enzymes are distributed over all twelve potato chromosomes. there is a concentration of ss on chromosome and many genes encoding bams are located on chromosome susy and isa encoding genes are distributed across different chromosomes. an interesting finding was the discovery of two pgm isoforms pgm and pgm which are located on chromosome and respectively. the sequence identity between both transcripts is %, but the corresponding genes differ significantly in their non-coding regions showing only 59% sequence similarity. however, the structure of both genes appears to be conserved. the pgm isoform has not been predicted by the pgsc or itag and was identified by blasting the transcript sequence sotub07g. against the scaffold sequences. investigating the tomato genome available on the sol genomics website for pgm genes revealed that only one locus is present which is localized on chromosome therefore, it is conceivable that the pgm gene on chromosome is the result of a recent gene duplication event, however this needs to be further investigated by bioinformatics analysis. identification of suitable microarray identifiers to investigate gene expression two oligonucleotide-based microarray platforms are available for global gene expression analysis in potato. the poci array was designed in 4x44k format based on a collection of expressed sequence tags while the 8x60k microarray is based on predicted transcript sequences of the dm potato genome by the pgsc. in this study, experimental data of both microarray designs were used. the prerequisite for the comparative expression analysis was the identification of suitable microarray oligonucleotides matching the transcript of interest, particularly in case of the poci platform. therefore, prior to expression analysis, oligonucleotide binding accuracy to the target genes was assessed. to this end, transcript and genomic sequences of starch genes were blasted against the poci database and resulting est sequences were aligned to the genomic sequence to allow for assessment of their corresponding oligonucleotide binding capacities to the transcript. oligonucleotides matching the reference sequence with 85% or more identity were considered for the analysis of expression profiles. due to the lack of matching est-sequences or to binding of the corresponding oligonucleotides within predicted introns, no suitable oligonucleotides were found for amy3-like, agps, agps tpt, tpt-like, gpt, all bam isoforms, bam bam pgi-like and ppgm in the poci platform. since oligonucleotide sequences of the 8x60k microarray were deduced from predicted transcript sequences of the dm genome, they perfectly match the corresponding transcript available at the spud db website. in these cases the position of the oligo within the gene was assessed to rule out that the binding site is within a putative intron. oligonucleotide specificity was investigated by multiple sequence alignments. the high sequence similarity between the transcripts of some isoenzymes prevented the assignment of specific oligonucleotides discriminating the isoforms of isa sex4-like, sbe and bam and bam. additional file lists all identifiers from both platforms that met our criteria and that were considered for further analyses. identification of genes that are highly expressed in leaves or tubers for the gene expression analysis, samples taken from leaf and tuber tissues were selected from different microarray experiments. raw data files of the different samples were uploaded into the genespring. gx software and were normalized together. direct comparisons of gene expression were made within the individual platforms first. afterwards derived results were compared between the different platforms. to identify starch genes that are preferentially expressed in leaves or tubers, the fold-change between the mean relative expression value detected in leaf and tuber samples was calculated using the genespring. gx software and displayed in additional file for genes, whose expression was ascertainable in both microarray platforms, the log fold-change was calculated and depicted in fig.  we considered genes that were on average more than 10-fold overexpressed in one tissue to be tissue-specific. the comparison between the two array platforms revealed that several genes are specifically expressed in leaves or tubers, respectively. hence, a strong tuber-specific expression was detected for gpt and susy followed by sex and ss whereas bam, apl and amy were found to be highly expressed in leaves. fold-change differences between leaf and tuber samples were often greater in the 8x60k array than in the poci array but the tendency was similar. the only exception was gpt whose expression was unchanged between leaf and tuber samples hybridized onto the poci array but showed a -fold higher expression in leaves than in tubers in samples analyzed on the 8x60k array.fig. relative expression of starch genes in leaf vs. tuber tissue. fold-change values for individual genes between leaf and tuber samples were exported from genespring or calculated from fpkm values from the pgsc database. genes whose expression cannot be detected in either platform were excluded. light grey bars: values from 8x microarray, black bars: values from 4x44k poci array, dark grey bars: fpkm values to confirm our results, fpkm values of corresponding genes were downloaded from rna-sequencing data available on the spud db website and leaf and tuber samples were selected. ratios between leaf and tuber values were calculated and compared to the results from the microarray analyses. fold-change values of the rna-seq data compared well to the microarray data. thus, gpt and susy are highly tuber-specifically expressed genes. their expression was 20- to 1000-fold higher in tubers compared to leaves. leaf-specific expression of amy, apl and bam could also be confirmed by the rna-seq data. they were found to be 7-fold to 320-fold higher expressed in leaves than in tubers. verification of differential expression of selected genes was carried out by quantitative real-time pcr. as shown in fig.  tuber-specific expression was confirmed for susy gpt and ss as well as the leaf-specific expression of amy, apl and bam. in addition, we selected two genes, apl and lsf showing a similar expression in leaves and tubers in all three transcriptome platforms. again, qrt-pcr analysis confirmed the transcriptome data.fig. qrt-pcr analysis of selected starch metabolism genes in potato leaves and tubers. plants were grown in a greenhouse for 11 weeks until harvest and sampling. mean relative expression of four biological replicates normalized to ef1alpha is illustrated as dct-value of a susy b apl c gpt, d bam, e ss f amy, g lsf h apl error bars represent standard deviation selection of query genes for co-expression analysis the main goal of the co-expression analysis was to identify possible regulators of starch biosynthesis in potato tubers. therefore, the genes used as queries for the analysis were selected by two criteria; first, they had to be specifically expressed in the tuber and second, their expression pattern had to follow starch accumulation. the first criterion was fulfilled most strongly by gpt, susy sex ss and sbe. for the evaluation of the second criterion, increasing gene expression during tuber development was chosen. it is known that during tuberization the rate of starch biosynthesis increases significantly. therefore, genes involved in starch biosynthesis should be upregulated during this process. to identify these genes, microarray data from the tuber induction experiment described by ferreira et al. were inspected and the ratio of transcripts detected in small tubers vs. those measured in unswollen stolons were calculated and illustrated as log values. the highest up-regulation from stage to stage was seen for susy sbe gpt and lde. sex which was identified as specifically expressed in tubers, showed a pronounced down-regulation in the course of tuber development. therefore, susy sbe and gpt were chosen as query genes for the co-expression analysis.fig. relative changes in expression of starch genes during tuber development. given are log transformed fold-changes. data were taken from ferreira et al. co-regulation analysis to identify putative regulators of starch metabolism in potato tubers to identify possible regulators of starch biosynthesis in potato tubers, all valid microarray identifiers for each of the selected genes were used as queries in a pearson correlation search on all detected entities in both microarray platforms including all data sets. in addition, rna-seq data were also analyzed. a pearson correlation coefficient of was used as cut-off. within each platform, the overlap of entities co-expressed with all three query genes was determined using venn diagrams. the numbers of genes co-regulated with gpt, susy and sbe differed greatly between platforms ranging between entities in the poci array, for the rna-seq data set and in the 8x60k array. to compare the results from the different platforms, found entities were assigned to their corresponding pgsc gene identification number. this resulted in a list of different genes that were consistently co-expressed with gpt, susy and sbe besides the three query genes, five other starch genes, namely apl pho1a, ss ntt and gpt were among the co-expressed genes and were identified to be tuber-specifically expressed. functional categorization of the co-expressed genes revealed that twenty percent of the co-expressed genes encode known storage proteins like patatin and protease inhibitors.fig. overview of co-expression analysis. a-c venn-analysis of co-expressed entities with susy, sbe and gpt. co-expression analysis was conducted using a pcc cut-off of  < =r < =. a co-expressed entities in the poci microarray platform, b co-expressed entities in the 8x60k microarray platform, c co-expressed entities in the rna-sequencing data. d after conversion of the co-expressed entity lists to gene lists, the lists were compared and the common genes in all three lists were retrieved to identify possible transcriptional regulators of starch biosynthesis in potato tubers, we paid special attention to putative tfs. among those, tfs with homology to regulators of organogenesis from arabidopsis like petal loss, lateral organ boundaries, blade on petiole and lateral root primordium protein were found. furthermore, a wrky-type tf and a member of the plant-specific tify motif containing protein family tify5a, were co-expressed with the starch biosynthesis genes. to confirm the expression profiles, four putative tf as well as susy and gpt were selected for qrt-pcr analysis. the relative amount of the corresponding mrna was quantified in an independent set of samples representing four different stages of tuber development, namely unswollen stolons, swollen stolons, growing tubers and dormant tubers. the results were compared to microarray data derived from similar stages of tuber development. as shown in fig.  the results from qrt-pcr were generally comparable to the results from microarray analysis when considering similar stages of tuber development. with respect to susy slight differences between both techniques were observed. while its expression reached highest values in stage in the microarray, a maximum transcript amount of susy was seen in growing tubers in qrt-pcr. the expression profiles of lob followed those of susy in both setups. wrky as well as tify5a showed similar profiles in both platforms and correlated highly to the expression of gpt. one exception was the expression profile of ptl. while its expression was lower in growing and non-growing tubers as compared to stage in the microarray experiments, the mrna level increased steadily across all developmental stages in the qrt-pcr reaching its maximum in dormant tubers.fig. expression profiles of tuber-specific starch genes and co-expressed transcription factors. a gene expression calculated as fold-change relative to the value at stage in the microarray experiments. b qrt-pcr analysis of the same genes in independent samples. each value represents the mean of 3– biological replicates. error bars represent standard deviation. black bars: susy dark grey bars: gpt, grey bars: ptl, medium grey bars: tify5a, light grey bars: lob domain containing protein, white bars: wrky4 a pearson correlation matrix was constructed evaluating the similarity of the expression profiles determined by qrt-pcr. most pcc values were greater than indicating that selected genes were co-regulated in the samples analyzed by qrt-pcr. however, the pcc values were lower than in the global co-expression analysis which is most likely due to the decreased sample number. thus, the qrt-pcr analysis corroborated that expression profiles of tify5a, lob and wrky are similar to those of susy and gpt during tuber development. for ptl, the pccs calculated from qrt-pcr were low supporting the observation that the expression pattern of this gene in the samples used for qrt-pcr deviated from the microarray.table pearson correlation coefficients between starch genes and tfs based on qrt-pcr analysis results from the qrt-pcr analysis of starch genes and transcription factors were subjected to a pearson correlation analysis using microsoft excel. correlation coefficients with p ≤  are indicated in bold letters discussion genome-wide analysis of starch genes in potato reveals novel isoforms enzymes involved in starch metabolism often belong to gene families encoding several isoenzymes. to our knowledge, this work presents the first genome-wide analysis of starch genes in potato. a comprehensive blast search strategy complemented by motif discovery and comparison to known sequences from arabidopsis was applied aiming at the identification of all “starch gene” loci in potato. we found loci coding for starch metabolism related enzymes belonging to different enzyme classes. higher plants possess five gene classes encoding starch synthases, designated gbss and ss1-. in rice, two forms of gbss were identified and eight genes encoding the four ss classes. in the potato genome, we confirmed that gbss which has been reported previously to be responsible for amylose biosynthesis in the starch granule, is encoded by a single gene and is expressed higher in tubers than in leaves. a second transcript annotated as gbss in the pgsc database had previously been described to possess soluble ss activity and was found to be the closest potato homolog to arabidopsis ss and was therefore consequently designated as ss moreover, it was shown that ss plays only a minor role in starch biosynthesis in tubers which is in accordance with our expression analysis showing only a slightly higher expression in tubers compared to leaves although being upregulated during tuber development. in total, seven starch synthases were found in the potato genome most of which have been described in earlier studies. however, no studies have analyzed the roles of ss and ss in potato yet, but our gene expression data suggest a possible role for ss in potato tuber starch biosynthesis. in contrast, ss is expressed to similar levels in leaves and tubers and its expression was not found to change significantly during tuber development. thus, further analyses are necessary to investigate the function of these genes during starch biosynthesis in potato. a recent publication described the phylogenetic relationship of ss from different plant species, including potato, confirming the presence of a fifth class of ss. in addition the authors found maize ss to be highly expressed during the grain filling stage suggesting a role in starch biosynthesis, which is in agreement with our assumption. in this study, enzymes were designated regarding to their annotation in arabidopsis. in most cases, this was in accordance with isoform numeration of already described enzymes of potato. one exception concerns the numeration of isoforms within the sbe class where we identified four isoenzymes. two of them share a very high sequence similarity to each other and have been denoted as sbe and sbe due to their homology to arabidopsis sbe the deduced transcript sequences of these two genes, however, do not correspond to the previously published potato sbe sequences. the gene product designated sbe in the aforementioned studies corresponds to sbe in this study. it was described as the major sbe isoform in potato tubers and was found to play a role in starch granule formation. this is in accordance with the expression profile during tuber development and tissue preference discovered in this study. until now, only variants of two isoforms, sbe and sbe have been shown to act as branching enzymes in the amyloplast. the role of the two potato sbe paralogs identified in this study remains unclear. in arabidopsis, sbe has an effect on embryogenesis and is essential for plant growth and development. a direct implication of atsbe in starch metabolism is unacquainted. comparative microarray analysis revealed tissue-specific gene expression to identify tuber- and leaf-specifically expressed starch genes different microarray data sets were analyzed. to enable the analysis, specific microarray probes had to be assigned to the different starch genes and their respective isoforms. in general, our findings were in agreement with previously published gene expression analyses and showed a high reproducibility between the two microarray platforms. tissue-specific expression of enzyme isoforms was for example found for pho1a and pho1b. pho1b appeared to be preferentially expressed in leaves, while pho1a was expressed higher in tubers, which is in accordance with previous findings. in the case of agpase, most subunits are expressed slightly higher in tubers than in leaves according to our results. however, one isoform, namely apl was clearly expressed higher in leaves than in tubers. this is in contrast to findings from la cognata et al. who described tuber-specific expression of apl. the reliability of our results was confirmed by rna-seq data and by qrt-pcr using leaf and tuber samples. genes showing tuber-specific expression were susy sbe ss gpt and sex in contrast to the other tuber-specific isoforms, sex4-specific transcripts were not up-regulated during tuber development which is consistent with the proposed role of the enzyme in starch degradation. the activity of the main susy isoform in tubers, susy is connected to the onset of tuberization and correlates well with transcript and tuber starch accumulation in potato. accordingly, susy overexpression led to an increased starch content and higher tuber yield in potato plants supporting its suggested key role in starch metabolism. similarly, sbe and gpt expression have been linked to tuber development and the accompanying accumulation of starch. in this context, overexpression of gpt together with ntt resulted in increased tuber starch content and yield indicating that expression and activity of gpt are closely related. the similarity between the expression patterns of these enzymes strongly supports the assumption of a coordinated transcriptional regulation of genes within the same pathway. moreover, these examples confirm that enzymatic activity of susy and gpt nicely correlates with transcript accumulation and that accumulation of starch metabolic enzymes is controlled at the transcriptional level. however, in other species activity of starch metabolic enzymes was shown to be additionally regulated by post-translational mechanisms. for example, phosphorylation of susy isoforms was shown to influence sub-cellular localization and protein stability. activity of sbe isoforms was reported to be regulated by protein phosphorylation and redox state. co-expression analysis reveals candidate regulators of starch biosynthesis co-expression analysis has previously been described to be a suitable tool for the identification of co-regulated genes. assuming that proteins with regulatory functions have to be expressed at the same time or shortly before their target genes, the identification of candidate regulators should be possible by co-expression analysis. the great potential of this strategy has already been demonstrated in several studies including different plant species and tissues. one example is the identification of rice starch regulator by fu and xue in a co-expression analysis similar to the approach used in this study. rsr was found to be negatively co-expressed with rice starch synthesis genes and was experimentally verified as a modulator of starch gene expression. in this work, genes that were identified as being tuber-specifically expressed and exhibiting an expression pattern that coincides with starch biosynthesis in the potato tuber were used to search for potential transcriptional regulators, since they are so far not known. the number of genes identified to be co-expressed with susy gpt and sbe3differed between the two microarray platforms, and was about times higher in the 8x60k experiments than in those performed with the poci platform. one reason for this might be the sample selection of the 8x60k platform which basically consists of tuber samples in similar developmental stages while most samples taken from the 4x44k format were originally designed to reflect starch biosynthesis during tuber formation. therefore, expression profiles derived from experiments using the poci array were expected to be more specific with respect to the identification of putative regulators of starch biosynthesis in potato tubers. moreover, we reasoned that co-expression of a regulator with its target genes should occur in all situations. thus, candidate selection was made after comparing the results of the co-expression analyses of the three query genes in three different platforms each with many individual samples. eventually we identified genes that are consistently co-regulated with susy gpt and sbe inspection of co-expressed genes revealed a strong over-representation of genes involved in primary carbon metabolism and development as well as genes encoding storage proteins. tuber development and storage metabolism are known to be highly associated processes which strengthens the significance of the retrieved candidates. beside this, putative tfs co-expressed with the selected starch genes could be identified. they belong to different classes and none of them has been characterized in potato so far. clearly, there is a strong enrichment of tfs associated with developmental processes and organogenesis like bop lob, ptl and lrp. for ptl, a co-expression with susy and gpt in samples representing different tuber developmental stages could not be confirmed via qrt-pcr and ptl might therefore not be a good candidate for further analysis. the expression profiles obtained by qrt-pcr of the other three tf were in accordance with those of the microarray analysis. slight variations between qrt-pcr and microarray were found when comparing expression levels of susy or lob on “stage 5” and “grow” from the microarray to “swollen stolon” and “growing tuber” samples used for qrt-pcr. in the microarray, highest gene expression was seen on “stage 5”, while in the qrt-pcr expression peaked in growing tubers. nevertheless, an increasing expression level was always associated with tuber formation. a possible explanation for this disagreement might be slightly different developmental stages of the samples used for the analyses. for the microarray defined stages of tuber development were sampled, while for the qrt-pcr swollen stolons of different developmental stages were pooled. furthermore, the growing tubers for the microarray experiment were monitored by x-ray ct analysis to determine their growth velocity, while the tubers sampled for qrt-pcr were taken from plants during their growth period, assuming that the tubers were still growing. despite these small differences between different experiments, expression levels of lob, tify5a and wrky correlate well with susy and gpt. thus they might be interesting candidates for further analyses. in arabidopsis, bop and its close homolog bop regulate the expression of lob-domain containing proteins. lob expression has been found in the boundary regions between meristematic tissue and developing lateral organ primordia of the shoot apical meristem and the roots. a similar spatial expression is exhibited by lrp of arabidopsis which has been shown to be expressed in root primordia in early developmental stages. in maize the localization of lrp in lateral root primordia was confirmed and it was demonstrated that lrp expression was auxin-inducible. a link to auxin-signaling may also be established by the closest homolog of potato wrky in arabidopsis. based on sequence similarity, the closest homolog in arabidopsis is wrky which has been linked to auxin-signaling in root development. a role of auxin in tuber initiation has been suggested but a direct link to starch biosynthesis is missing. the expression patterns of these tfs suggest that there are interesting candidate genes which may directly or indirectly control starch biosynthesis and that more detailed investigation of their role is worthwhile.  in this study the complete inventory of starch metabolism genes and their genomic localization was described which will facilitate future examinations of the distinct functions of isoenzymes in this pathway. moreover, novel as far undescribed enzyme isoforms were identified whose characterization will shed more light on the mechanisms of starch biosynthesis and degradation in potato plants. comparative microarray analysis uncovered leaf- and tuber-specific starch gene isoforms. this finding suggests distinct regulatory mechanisms in transitory and storage starch metabolism. a co-expression analysis was conducted using tuber-specific genes aiming at the identification of regulators of starch biosynthesis in potato tubers. forty genes showed strong co-regulation in all platforms analyzed. among the co-expressed genes were many storage metabolism genes belonging to the starch biosynthesis pathway or storage proteins as well as tfs. none of the identified tfs had been described in potato yet, but many of their homologs in arabidopsis are known regulators of lateral organ development. we conclude that tuber development and tuber starch biosynthesis are highly connected pathways and consider it worthwhile to investigate the influence of the identified regulators on starch biosynthesis. 
moreover, mechanisms regulating the expression of starch genes in leaves and tubers remain elusive with regard to differences between transitory and storage starch metabolism, respectively. this study aimed at identifying and mapping the complete set of potato starch genes, and to study their expression pattern in leaves and tubers using different sets of transcriptome data. expression analysis of starch genes led to the identification of tissue-specific isoenzymes suggesting differences in the transcriptional regulation of starch metabolism between potato leaf and tuber tissues. comparative transcriptome analysis enabled the identification of tuber- and leaf-specific isoforms of starch genes. selection of genes predominantly expressed in developing potato tubers and exhibiting an expression pattern indicative for a role in starch biosynthesis enabled the identification of possible transcriptional regulators of tuber starch biosynthesis by co-expression analysis. starch metabolismgene expressionmicroarray analysissolanum tuberosumgene annotationco-expression analysishttp://dx.doi.org/ /501100001659deutsche forschungsgemeinschaftso1002/3-1sonnewald sophia http://dx.doi.org/ /501100005908bundesministerium für ernährung und landwirtschaft22032711sonnewald uwe issue-copyright-statement© the author 2017 this finding suggests distinct regulatory mechanisms in transitory and storage starch metabolism. moreover, we wanted to uncover transcription factors co-regulated with starch accumulation in tubers in order to get insight into the regulation of starch metabolism. this study provides the annotation of the complete set of starch metabolic genes in potato plants and their genomic localizations. despite its importance, a description of the complete inventory of genes involved in starch metabolism and their genome organization in potato plants is still missing. we identified genomic loci encoding enzymes involved in starch metabolism. their analysis will help to elucidate mechanisms of starch biosynthesis and degradation. putative regulatory proteins of starch biosynthesis in potato tubers have been identified by co-expression and their expression was verified by quantitative rt-pcr. novel, so far undescribed, enzyme isoforms were revealed. starch is the principle constituent of potato tubers and is of considerable importance for food and non-food applications.
 starch is the principle constituent of potato tubers and is of considerable importance for food and non-food applications. its metabolism has been subject of extensive research over the past decades. despite its importance, a description of the complete inventory of genes involved in starch metabolism and their genome organization in potato plants is still missing. moreover, mechanisms regulating the expression of starch genes in leaves and tubers remain elusive with regard to differences between transitory and storage starch metabolism, respectively. this study aimed at identifying and mapping the complete set of potato starch genes, and to study their expression pattern in leaves and tubers using different sets of transcriptome data. moreover, we wanted to uncover transcription factors co-regulated with starch accumulation in tubers in order to get insight into the regulation of starch metabolism.  we identified genomic loci encoding enzymes involved in starch metabolism. novel isoforms of many enzymes were found. their analysis will help to elucidate mechanisms of starch biosynthesis and degradation. expression analysis of starch genes led to the identification of tissue-specific isoenzymes suggesting differences in the transcriptional regulation of starch metabolism between potato leaf and tuber tissues. selection of genes predominantly expressed in developing potato tubers and exhibiting an expression pattern indicative for a role in starch biosynthesis enabled the identification of possible transcriptional regulators of tuber starch biosynthesis by co-expression analysis.  this study provides the annotation of the complete set of starch metabolic genes in potato plants and their genomic localizations. novel, so far undescribed, enzyme isoforms were revealed. comparative transcriptome analysis enabled the identification of tuber- and leaf-specific isoforms of starch genes. this finding suggests distinct regulatory mechanisms in transitory and storage starch metabolism. putative regulatory proteins of starch biosynthesis in potato tubers have been identified by co-expression and their expression was verified by quantitative rt-pcr. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords starch metabolismgene expressionmicroarray analysissolanum tuberosumgene annotationco-expression analysishttp://dx.doi.org/ /501100001659deutsche forschungsgemeinschaftso1002/3-1sonnewald sophia http://dx.doi.org/ /501100005908bundesministerium für ernährung und landwirtschaft22032711sonnewald uwe issue-copyright-statement© the author 2017 