BACKGROUND
nicotiana tabacum  is a member of the solanaceae family, which also includes tomato  and potato , amongst other commercially important crop plants.
it was originally studied due to its economic importance but came to the fore of plant biology when it was the first plant to be genetically modified in  <dig>  <cit> .
tobacco is an allotetraploid  derived from the interspecific hybridisation of two progenitor genomes; a maternal 's' genome, originating from an ancestor of n. sylvestris, and a paternal 't' genome, most likely originating from a lineage of n. tomentosiformis  <cit> .
it has a genome of approximately  <dig> gb  <cit> , but despite its commercial and scientific importance relatively little is known about the plant's genome sequence.
recent efforts by the tobacco genome inititative  have increased the sequence information available for the transcriptionally active regions of the tobacco genome.
some of this information is in the form of expressed sequence tags ; short, single pass sequence reads derived from complementary dna  libraries, whilst others are methyl filtered genome space sequence reads .
ests provide an insight into transcriptionally active genes in a biological sample under a given set of conditions.
est sequencing is relatively expensive and time consuming.
microarrays, however, provide a faster less costly alternative for measuring gene expression for thousands of genes simultaneously that can be more easily and reproducibly applied across a broad range of conditions or treatments to identify genes showing specific expression patterns or responses.
it should be noted that microarray measurements provide an estimate of the steady state level of transcripts.
they do not capture all of the post-transcriptional or post-translational regulation of genes and as such do not necessarily reflect the expression of a gene in terms of functional protein produced.
microarrays have been used in other plant species and organisms such as insects and mammals to capture the variation in gene expression in different tissues throughout development  <cit> .
these studies show that organs can be differentiated based on their transcriptional signatures, with sets of genes involved in specific biological processes being expressed only in the relevant tissues.
such data sets provide researchers with information on their gene of interest's expression over a broad range of conditions.
inference of gene function based on co-expression with other annotated genes is a key component of functional genomics.
community-wide projects, such as atgenexpress , and the development of centralised microarray service providers and data repositories, such as the nottingham arabidopsis stock centre , have resulted in a large amount of publicly available expression data for the model plant arabidopsis thaliana.
development of this resource has enabled comparative analyses to be carried out leading to the identification of strongly co-expressed genes and aiding the elucidation of biochemical pathways for example  <cit> .
associated with the broad availability of such data has been the development of both stand-alone and web-based tools to allow researchers to fully interrogate the expression data sets to further their own research  <cit> .
transcription factors play a key role in regulating gene expression and have been identified by comprehensive analyses in a number of plant species  <cit> .
methyl filtered genomic sequence reads have been used to identify 80-90% of the transcription factors in tobacco  <cit> .
functional genomics is based on a guilt-by-association approach, whereby co-expression of a gene of unknown function with other genes known to be involved in a common biochemical or signalling pathway may suggest that the unknown gene is also involved in this pathway.
co-expression of genes can also be used to infer putative transcriptional networks  <cit> .
the identification of transcription factors that act as regulators of specific biochemical or signalling pathways potentially provide prime targets for manipulation to alter the agronomic or biochemical characteristics of the plant.
such manipulation could prove very useful in projects related to secondary metabolite or biofuel production, or in the case of tobacco leaves, reducing the level of metabolites that give rise to toxicants in cigarette smoke.
this study describes the generation of a custom affymetrix expression array for tobacco and its use in developing the tobacco expression atlas .
tobea is a map of gene expression from multiple tissues sampled throughout the life cycle of the tobacco plant and is intended to be used as a reference data set for plant researchers.
the expression data is freely available via the solanaceae genomics network , a web based genomic resource for plants of the solanaceae family .
several tools have also been developed to facilitate access to information on researcher's genes of interest.
RESULTS
generation of a microarray for tobacco
in order to enable gene expression studies in tobacco, generation of a microarray based on nicotiana tabacum sequences was undertaken.
currently genbank contains over  <dig> tobacco ests, however, prior to the initiation of this project less than  <dig> sequences were publicly available for tobacco.
to increase the quantity of available sequence data  <dig> cdna libraries were generated from multiple tissue types of several tobacco varieties, although there was a focus towards libraries originating from variety k <dig> and leaf samples.
table  <dig> summarises the variety and tissue of origin, along with the number of ests generated from each of the libraries.
table showing the tissue type and tobacco variety sampled in generating  <dig> est libraries.
the number of ests in each library is shown.
one of two methods was applied to minimise redundancy within each library and maximise the number of unique ests generated.
five of the libraries  underwent normalisation prior to sequencing .
this generated  <dig> ests, of which  <dig>  could successfully be sorted into their library of origin based on library specific sequence tags.
despite lacking tags, the remaining  <dig> sequences still contained valuable sequence information so were maintained as a generic n. tabacum leaf est library .
the remaining  <dig> libraries  underwent virtual subtraction to reduce redundancy  <cit> .
the virtual subtraction libraries were generated as part of the european sequencing of tobacco  project and were included in the tobacco unigene set generated by this effort http://www.estobacco.info.
the  <dig> ests generated by this study, along with another  <dig> n. tabacum sequences available at the time were assembled into a set of  <dig> tobacco unigenes.
<dig> of the  <dig> sequences assembled into  <dig> contigs, with the remaining  <dig> singleton sequences completing the unigene set, which ranged in length from  <dig> bp to  <dig> bp .
functional annotations for the unigenes were taken from the best blastx hit of arabidopsis thaliana proteins , and/or from the blastx hit of non-redundant proteins from genbank using the program blast2go.
in total  <dig>  of the unigenes were annotated by either method .
the affymetrix custom design service was used to produce a tobacco expression array based on the tobacco unigene sequences described above.
of the  <dig> sequences submitted,  <dig> were omitted from the array as suitable probes could not be identified.
standard hybridisation and polya controls were included on the array together with four species-specific maintenance genes.
a small proportion of the sequences were represented by  <dig> probe sets, resulting in a total of  <dig> probe sets present on the array.
temporal and spatial changes in tobacco gene expression
temporal and spatial regulation of gene expression plays a critical role in shaping plant growth and development and considerable variation in gene expression at these levels has been demonstrated in several plant species  <cit> .
understanding where and when expression occurs provides a valuable insight into gene function and the functioning of the plant as a whole.
using the custom designed affymetrix expression array, this variation was captured for tobacco genes by measuring gene expression in different tissue types at various stages throughout plant development .
for ease of representation each of the  <dig> conditions in the tobacco expression atlas  was given a tissue type index  number, with samples from the same tissue/organ grouped together and then ordered temporally based on developmental stage .
data were pre-processed with the mas <dig> algorithm to allow the future addition of further tissue types to the tobea database  <cit> .
based on the mas <dig> flag values, genes were considered expressed if they were called as present or marginal in at least four out of the five biological replicates for at least one tissue type.
on this basis genes corresponding to  <dig> of the  <dig> probe sets on the array were expressed.
detection of expression for 76% of the probesets on the array was considered reasonable as tobea did not comprehensively cover all of the conditions and varietal differences represented in the est libraries used to generate the sequences that the array was designed against.
the detection level is also not much lower than that shown previously in a larger study in arabidopsis  <cit> .
of the expressed genes,  <dig> showed a significant difference in expression between tissues, based on a one way anova  following the removal of non-changing genes .
the number of differentially expressed genes identified suggested that expression varied significantly between tobacco tissues and throughout development for the majority of genes in the plant.
tissues/organs in other plant species can be distinguished based on their transcriptional signature  <cit> .
principal components analysis  was carried out on the tobea data to determine the relationship between tobacco tissues.
figure  <dig> shows the individual tobea microarrays plotted within the first three principal components .
for each tissue type, the five biological replicate samples clustered closely together, suggesting that they were a reliable representation of the transcriptional profile for their respective tissue .
the  <dig> different tissue types could be broadly sub-divided into  <dig> clusters based on their position within pc space .
samples from imbibed seed  showed distinct separation from the main body of samples along pc <dig> .
the root  and cotyledon/leaf  samples were also separated along pc <dig>  with the remaining tissue types falling between these groups .
this suggested that pc <dig> was representative of some of the spatial variation in tobacco gene expression.
some tissue types, such as roots , could not be distinguished based on the developmental stage, however, other tissue types did show a developmental separation along pcs  <dig> and  <dig> .
this was apparent for the transition from floral apex to floral buds in cluster viii .
greater separation in pc space for a developmental series was shown by leaves.
cotyledon and young shoot samples  along with young leaf samples  showed clear separation from mature and senescing leaf samples  along pcs  <dig> and  <dig> .
this showed a clear distinction in transcriptional signature between young and old leaves.
gene expression changes during tobacco leaf development
to investigate the underlying cause of the separation between young and mature/senescing leaf material, genes were identified that showed differential expression across the 'leaf' samples .
genes were called differentially expressed if they showed significant variation  and a minimum of five-fold change in expression for at least one pairwise comparison between young  and mature  leaf samples.
the  <dig> genes identified on this basis were clustered by k-means into  <dig> groups as shown in figure  <dig>  two basic responses were represented by the  <dig> clusters; genes showing relatively higher expression in younger samples  and genes showing relatively higher expression in older samples .
clusters were tested for over-representation of gene functions, based on the annotations borrowed from the best arabidopsis blast hit.
when genes in clusters l <dig> and l <dig> were combined, the gene ontology  analysis showed over-representation of multiple terms relating to photosynthesis and cell cycle, or cell growth and expansion .
a combined list of the genes in clusters l <dig> and l <dig> showed over-representation of go terms relating to senescence related responses, including break down and re-mobilisation of various metabolites, hormone responses and stress responses .
this suggested that there was a clear distinction between gene expression in young and old leaf material, with the former showing greater activity in photosynthesis, growth and expansion and the latter undergoing senescence.
although the four clusters were defined by two basic responses, normalisation of the data across all of the tissues in tobea allowed further distinction of the clusters.
for instance, genes in clusters l <dig> and l <dig> both showed relatively higher expression in younger leaves than older leaves, however, in the context of other tissues in the expression atlas, cluster l <dig> genes were relatively highly expressed in young leaf samples, whereas cluster l <dig> genes showed relatively low expression in older leaf samples .
go analysis of the genes in cluster l <dig> principally showed terms associated with photosynthesis, whereas genes in cluster l <dig> showed terms related to cell cycle or cell growth and expansion .
together, this suggested that the differences between young and old leaf tissues can be described as increased photosynthetic activity in younger samples and reduced cell division and growth along with increased senescence in older leaf tissues.
this clearly shows the benefit of considering changes in gene expression in the context of all of the other tissue types in the tobea database and not just between a limited subset of tissue types.
tissue specific tobacco gene expression
the above analyses demonstrated that many tobacco genes show changes in expression between different tissues.
to test for genes that are only expressed in specific tissues, all of the expressed genes were identified for each tissue type.
for this analysis only genes flagged as present or marginal in all  <dig> biological replicates of at least one developmental stage within a tissue type were considered expressed.
figure  <dig> shows a venn diagram for each of the six tissue types represented in the tobea database, with the genes identified as expressed in each tissue compared to a combined list of all those identified as expressed in the other tissues.
go analysis was carried out on genes identified as expressed specifically in a single tissue type .
the  <dig> genes identified as specifically expressed in imbibed seed samples  showed over-representation of terms associated with seed development and nutrient storage as well as those responsive to water and high light intensity, all processes/responses that are likely to be well represented in an imbibed seed .
similarly, genes specifically expressed in root samples  showed over-representation of terms related to nutrient uptake, transport and toxin or defence responses.
those specifically expressed in leaf samples  showed over-representation of genes involved in photosynthesis .
additionally, genes specifically expressed in stem , shoot apices  and flower buds/flowers  showed over-representation of genes associated with microtubule development, implicated in the regulation of stem growth  <cit> , chromosome segregation/dna metabolism and floral organ development respectively .
co-expression of tobacco genes
the set of  <dig> genes showing differential expression across the tobea samples were analysed for co-expression.
figure  <dig> shows a subset of  <dig> clusters from a set of  <dig> k-means clusters .
go analysis was carried out on the  <dig> clusters and many of the identified biological processes could be related to the tissue types they showed expression in.
for example genes in cluster k <dig> showed relatively high expression in imbibed seed samples  and an over-representation of genes involved in seed development and nutrient storage .
indeed 50% of the genes in cluster k <dig> were among the  <dig> identified as specifically expressed in imbibed seed.
similarly genes in cluster k <dig> showed relatively high expression in buds and flowers , and genes in clusters k <dig> showed relatively high expression in young leaves  and these  <dig> clusters showed over-representation of genes related to floral development and photosynthesis respectively .
another example of expression of genes involved in relevant processes for the respective tissue is offered by cluster k <dig>  these genes showed high expression in both root and stem samples and over-representation of genes involved in xylem and phloem development .
clusters k13/k <dig> and k4/k8/k <dig> showed relatively high and relatively low levels of gene expression in mature and senescing leaf samples respectively .
the former set of clusters showed over-representation of genes involved in senescence related processes and the latter for genes involved in cell cycle and growth .
together with the results for cluster k <dig>  this supported the suggestion that transcriptional signature differences between young and old leaf tissues was based on genes involved in photosynthesis, cell division and growth, or senescence.
analysis of tobacco transcription factor expression patterns
transcription factors  play an important role in the regulation of gene expression.
comprehensive surveys of the tfs present in several other plant species have been undertaken  <cit> .
although complete sequence coverage is not available for the tobacco genome, the gsr sequence data generated by the tgi allowed the identification of  <dig> tfs in n. tabacum as part of the tobfac database  <cit> .
a blastn search was used to map these tfs to the tobacco unigenes assembled during this study .
the  <dig> tfs were mapped to  <dig> unique unigenes, of which  <dig> were represented by at least one probe set  on the microarray.
of the tobfac tfs mapped to probe sets on the array, 87% were identified as expressed in at least one tissue.
figure  <dig> shows the number and percentage of tobacco microarray probe sets representing tfs for each of the tobfac families scored as expressed.
for the majority of the tf families over 60% of their members were scored as expressed .
the exceptions to this were the shi related sequence  and cpp families, where only 50% and 0% were expressed respectively .
however, it should be noted that both these families were represented by two or less probe sets .
the ethylene response factors  are one of the largest tf families in tobacco with over  <dig> members  <cit>  and are represented by  <dig> probesets on the tobacco microarray.
the erf family did not show any strong trend in tissue specificity within tobea, with a large number being expressed in all of the different tissue types .
the erfs are divided into  <dig> subgroups, and a high percentage of group ix tobacco erfs were previously shown to be induced in response to methyl jasmonate  treatment  <cit> .
the group ix erfs were represented by  <dig> probe sets on the tobacco microarray.
compared to the family as a whole, the group ix erfs showed fewer members expressed in all other tissues  aside from roots, where  <dig> % of them were scored as expressed.
meja treatment has been shown to inhibit root growth in arabidopsis  <cit>  and tobacco  <cit>  and it may be that the induction of these root expressed group ix erfs by meja plays an important role in the inhibition of growth.
identification of putative transcriptional networks
although expression could be measured for a majority of tfs across a range of different tissues, this does not mean that their expression levels were similar between tissues.
correlation of expression level across different conditions provides a useful tool for functional genomics.
the program biolayout 3d was used to generate gene networks based on co-expression of tfs across all of the arrays in tobea  <cit> .
individual probe sets for tfs are represented by nodes in the network and the edges between nodes represent correlated gene expression.
of the  <dig> probe sets representing tfs on the array,  <dig> were placed into  <dig> networks.
figure  <dig> shows an example of one of the networks generated including orthologs for  <dig> mads-box tfs and one yabby tf involved in floral meristem identity in other plant species .
it should be noted that an edge between two nodes does not necessarily indicate a causal interaction between these nodes as it could equally be interpreted as co-regulated expression of both nodes by a common regulator.
however, several interactions suggested by the tobacco floral identity gene network were consistent with the gene regulatory network proposed for arabidopsis .
for example putative interactions between apetala <dig>  and ap <dig>  between ap <dig> and agamous  and both of these genes with pistillata  in tobacco are supported by results in arabidopsis  <cit> .
some interactions shown in arabidopsis were not represented in the tobacco tf network, such as a positive interaction between ap <dig> and pi and a negative interaction between ag and ap <dig>  failure to see the latter interaction is not surprising as the network is based on positive correlation.
the tf network also suggests a positive interaction between ap <dig> and agamous like  <dig> , whereas agl <dig> is reportedly repressed by ap <dig> in arabidopsis .
this suggests a different relationship may exist between these genes in tobacco compared to arabidopsis.
two putative tobacco agl <dig> orthologs were identified, so it was possible that only one had lost the repression by ap <dig>  however, the other agl <dig> ortholog was placed in a separate small network along with another ap <dig> ortholog , suggesting that both of the tobacco agl <dig> orthologs were not repressed by ap <dig> .
the tobacco tf network appeared to consist of two distinct sub-clusters, one including agl <dig> and ap <dig> orthologs and the other including orthologs for ap <dig>  pi, ag, crabs claw , sepallata <dig>  and seedstick  .
this network structure is consistent with the former two genes being involved earlier on during floral determination, whereas the remaining genes have been implicated later in floral development and floral organ identity  <cit> .
development of web-based tools for tobea
the tobacco microarray was designed from a unigene assembly based on  <dig> sequences.
since the assembly was carried out there has been a significant increase in the sequence data available for tobacco, with over  <dig> k ests currently available at genbank.
a more recent set of  <dig> unigenes was assembled from  <dig> n. tabacum sequences by the sgn.
the sgn also houses nicotianacyc, a database linking the tobacco unigenes to the metacyc biochemical pathways  <cit> .
to benefit from the greater genomic coverage and annotation information, unigenes present on the microarray were mapped to the current sgn unigene assembly .
<dig>  of the probe sets on the microarray could be mapped to  <dig>  of the sgn unigenes, indicating approximately 38% coverage of the sgn unigene build by the microarray.
approximately 81% of the mapped sgn unigenes were represented by a unique probe set, and over 99% by  <dig> probe sets or less.
information of the temporal and spatial expression pattern of genes, along with co-expression across a broad range of conditions can be very useful in inferring function for unknown genes.
in order to make the information contained within tobea more accessible to other researchers and enable them to place information on their gene of interest within the context of its expression information, it was decided to integrate the tobea expression data into the sgn website.
figure  <dig> shows some sample screen shots of the website, where users can access expression information on their gene of interest.
the sgn expression data module  allows users to view the expression level of their unigene across the different samples included in tobea .
the sedm also allows users to identify other sgn unigenes that show correlated expression across the tobea microarray data set .
discussion
recent efforts, such as the tgi, have massively increased the sequence resources available for n. tabacum.
however, the  <dig> ests generated by this study still represent a significant contribution to the total number of ests available for tobacco and will prove very useful in applications such as the detection of varietal polymorphisms or gene model predictions.
along with  <dig> other available sequences, these ests were assembled into a set of  <dig> unigenes and used to design a custom affymetrix expression microarray for tobacco.
affymetrix chips are a proven and widely accepted technology and the design service has been used successfully to generate a large number of custom chips for other research applications http://www.affymetrix.com.
the resultant microarray was used to capture the temporal and spatial changes in gene expression throughout the lifecycle of the tobacco plant.
considerable variation in gene expression was shown across the different tissues within tobea, with the transcriptional signatures revealing relationships between samples.
as observed for arabidopsis the transcriptional profile of imbibed seed showed the greatest difference to all other samples causing them to be distinctly removed in principal component space  <cit> .
pca also showed a broad distinction between samples originating from root and shoot tissues.
studies in other plants have shown that the complement of genes expressed in a tissue is related to the biological function that tissue performs  <cit> .
analysis of the go annotations shows that this is also true in tobacco.
as well as spatial changes, temporal changes in gene expression were apparent over developmental series in the same tissues.
this was more apparent for some tissues than others.
for example pca showed little separation between young and mature roots.
there was, however, a tendency for stronger expression of genes related to cell division, growth and expansion  in younger tissues and stronger expression of senescence related genes in older tissues.
on this basis there was a clear separation between young and old leaf samples, consistent with findings for arabidopsis  <cit> .
consistency of the results contained within tobea with previous results in other plants suggests that it is a high quality expression data set and a good representation of the spatial and temporal changes in gene expression throughout the lifecycle of the tobacco plant.
together with close clustering of independent replicate samples this also indicates that the microarray can be used to reliably and reproducibly measure gene expression in tobacco.
since the study was initiated larger unigene sets have been assembled for tobacco from the increased amount of sequence data available.
these new unigene assemblies likely represent greater coverage of the tobacco genome.
mapping of the microarray probe sets to the current unigene build from the sgn showed that the microarray still represents around 38% coverage of tobacco genes.
mapping of the probe sets to any future unigene assemblies will be maintained to ensure that the annotation information available for the array remains up to date.
as demonstrated by tobfac, the tobacco gsr sequence data provides the potential to identify genes that are not represented in the available est libraries  <cit> .
this is most likely the case for rare transcripts or those showing temporal, spatial, or alternative forms of regulation that is not captured in the conditions sampled by the current est libraries.
in future assemblies, the incorporation of sequence data such as gsrs with est sequences has the potential to provide more comprehensive coverage of the genes present in the tobacco genome than the est data alone.
the microarray represents a very valuable tool for research in tobacco, allowing comprehensive expression studies to be carried out in the plant.
to maximise the benefit of this the array has been made publicly available for use via the affymetrix service at nasc http://affymetrix.arabidopsis.info.
the microarray might also provide a very useful tool to researchers in closely related species, in particular other nicotiana species, via cross-species hybridisations  <cit> .
tobea provides a useful reference data set to any future experiments carried out using the microarray, allowing a more detailed characterisation of the differentially expressed genes identified.
to facilitate use of the information contained in tobea, a set of web-based tools were developed.
these tools currently allow researchers to view the expression level of their gene of interest across the different tissues in the atlas and identify other genes that show highly correlated expression via the sgn website.
this will enable individual users to carry out functional genomic analysis allowing the annotation of the sgn unigenes to be updated by experts from all different fields of biology via the locus editor feature of the sgn website.
as further experiments are carried out using the tobacco microarray, the data will be uploaded into the sgn expression database sedm.
the sedm can store results for any solanaceae species, and is capable of storing data sets from both microarray and direct sequencing studies.
enabling users to upload expression data to the sgn expression database means it has the potential to become a very useful resource for all researchers in the solenaceae.
the value of tobea is demonstrated by the study of specific sets of genes such as transcription factors.
due to their regulatory role, tfs represent good targets for gross modifications of biochemical or signalling pathways for various biotechnological applications  <cit> .
over 30% of the tfs identified by rushton et al.,  <cit>  in a genome scale survey of tobacco could be mapped to unigenes on the microarray.
simple correlation of tf expression levels across the tissues in tobea enabled the construction of a gene network involved in floral determination and floral organ identity.
several of the connections observed in this network were consistent with known interactions from other plant species.
extending this type of network analysis to include genes beyond tfs could allow the identification of putative transcriptional networks for specific metabolic or signalling pathways.
this analysis could identify regulatory targets to enable the manipulation of these pathways in efforts to increase or decrease the production of various secondary metabolites in tobacco.
CONCLUSIONS
an affymetrix microarray was developed that allows reliable and reproducible measurement of gene expression in tobacco.
this array is available for public use via the nasc affymetrix service.
the array was used to produce the tobacco expression atlas , which provides a snap-shot of gene expression from multiple tissues throughout the lifecycle of the tobacco plant.
a set of web based tools were developed in order to facilitate public access to this data via the sgn web site http://solgenomics.net/.
it is intended that the database will be extended in future with the addition of further expression datasets, not only from tobacco but other plants of the solanaceae family.
the sgn welcomes the donation of such data from the research community.
tobea represents a valuable resource for functional genomic analysis in plants and has potential application in research leading to biotechnological and agronomic improvements to tobacco as well as related species.

 nicotiana tabacum is a member of the solanaceae family, which also includes tomato and potato, amongst other commercially important crop plants. it was originally studied due to its economic importance but came to the fore of plant biology when it was the first plant to be genetically modified in. tobacco is an allotetraploid derived from the interspecific hybridisation of two progenitor genomes; a maternal's' genome, originating from an ancestor of n. sylvestris, and a paternal't' genome, most likely originating from a lineage of n. tomentosiformis. it has a genome of approximately gb, but despite its commercial and scientific importance relatively little is known about the plant's genome sequence. recent efforts by the tobacco genome inititative have increased the sequence information available for the transcriptionally active regions of the tobacco genome. some of this information is in the form of expressed sequence tags; short, single pass sequence reads derived from complementary dna libraries, whilst others are methyl filtered genome space sequence reads. ests provide an insight into transcriptionally active genes in a biological sample under a given set of conditions. est sequencing is relatively expensive and time consuming. microarrays, however, provide a faster less costly alternative for measuring gene expression for thousands of genes simultaneously that can be more easily and reproducibly applied across a broad range of conditions or treatments to identify genes showing specific expression patterns or responses. it should be noted that microarray measurements provide an estimate of the steady state level of transcripts. they do not capture all of the post-transcriptional or post-translational regulation of genes and as such do not necessarily reflect the expression of a gene in terms of functional protein produced. microarrays have been used in other plant species and organisms such as insects and mammals to capture the variation in gene expression in different tissues throughout development. these studies show that organs can be differentiated based on their transcriptional signatures, with sets of genes involved in specific biological processes being expressed only in the relevant tissues. such data sets provide researchers with information on their gene of interest's expression over a broad range of conditions. inference of gene function based on co-expression with other annotated genes is a key component of functional genomics. community-wide projects, such as atgenexpress, and the development of centralised microarray service providers and data repositories, such as the nottingham arabidopsis stock centre, have resulted in a large amount of publicly available expression data for the model plant arabidopsis thaliana. development of this resource has enabled comparative analyses to be carried out leading to the identification of strongly co-expressed genes and aiding the elucidation of biochemical pathways for example. associated with the broad availability of such data has been the development of both stand-alone and web-based tools to allow researchers to fully interrogate the expression data sets to further their own research. transcription factors play a key role in regulating gene expression and have been identified by comprehensive analyses in a number of plant species. methyl filtered genomic sequence reads have been used to identify 80-90% of the transcription factors in tobacco. functional genomics is based on a guilt-by-association approach, whereby co-expression of a gene of unknown function with other genes known to be involved in a common biochemical or signalling pathway may suggest that the unknown gene is also involved in this pathway. co-expression of genes can also be used to infer putative transcriptional networks. the identification of transcription factors that act as regulators of specific biochemical or signalling pathways potentially provide prime targets for manipulation to alter the agronomic or biochemical characteristics of the plant. such manipulation could prove very useful in projects related to secondary metabolite or biofuel production, or in the case of tobacco leaves, reducing the level of metabolites that give rise to toxicants in cigarette smoke. this study describes the generation of a custom affymetrix expression array for tobacco and its use in developing the tobacco expression atlas. tobea is a map of gene expression from multiple tissues sampled throughout the life cycle of the tobacco plant and is intended to be used as a reference data set for plant researchers. the expression data is freely available via the solanaceae genomics network, a web based genomic resource for plants of the solanaceae family. several tools have also been developed to facilitate access to information on researcher's genes of interest.  generation of a microarray for tobacco in order to enable gene expression studies in tobacco, generation of a microarray based on nicotiana tabacum sequences was undertaken. currently genbank contains over tobacco ests, however, prior to the initiation of this project less than sequences were publicly available for tobacco. to increase the quantity of available sequence data cdna libraries were generated from multiple tissue types of several tobacco varieties, although there was a focus towards libraries originating from variety k and leaf samples. table summarises the variety and tissue of origin, along with the number of ests generated from each of the libraries. table showing the tissue type and tobacco variety sampled in generating est libraries. the number of ests in each library is shown. one of two methods was applied to minimise redundancy within each library and maximise the number of unique ests generated. five of the libraries underwent normalisation prior to sequencing. this generated ests, of which could successfully be sorted into their library of origin based on library specific sequence tags. despite lacking tags, the remaining sequences still contained valuable sequence information so were maintained as a generic n. tabacum leaf est library. the remaining libraries underwent virtual subtraction to reduce redundancy. the virtual subtraction libraries were generated as part of the european sequencing of tobacco project and were included in the tobacco unigene set generated by this effort http://www.estobacco.info. the ests generated by this study, along with another n. tabacum sequences available at the time were assembled into a set of tobacco unigenes. of the sequences assembled into contigs, with the remaining singleton sequences completing the unigene set, which ranged in length from bp to bp. functional annotations for the unigenes were taken from the best blastx hit of arabidopsis thaliana proteins, and/or from the blastx hit of non-redundant proteins from genbank using the program blast2go. in total of the unigenes were annotated by either method. the affymetrix custom design service was used to produce a tobacco expression array based on the tobacco unigene sequences described above. of the sequences submitted, were omitted from the array as suitable probes could not be identified. standard hybridisation and polya controls were included on the array together with four species-specific maintenance genes. a small proportion of the sequences were represented by probe sets, resulting in a total of probe sets present on the array. temporal and spatial changes in tobacco gene expression temporal and spatial regulation of gene expression plays a critical role in shaping plant growth and development and considerable variation in gene expression at these levels has been demonstrated in several plant species. understanding where and when expression occurs provides a valuable insight into gene function and the functioning of the plant as a whole. using the custom designed affymetrix expression array, this variation was captured for tobacco genes by measuring gene expression in different tissue types at various stages throughout plant development. for ease of representation each of the conditions in the tobacco expression atlas was given a tissue type index number, with samples from the same tissue/organ grouped together and then ordered temporally based on developmental stage. data were pre-processed with the mas algorithm to allow the future addition of further tissue types to the tobea database. based on the mas flag values, genes were considered expressed if they were called as present or marginal in at least four out of the five biological replicates for at least one tissue type. on this basis genes corresponding to of the probe sets on the array were expressed. detection of expression for 76% of the probesets on the array was considered reasonable as tobea did not comprehensively cover all of the conditions and varietal differences represented in the est libraries used to generate the sequences that the array was designed against. the detection level is also not much lower than that shown previously in a larger study in arabidopsis. of the expressed genes, showed a significant difference in expression between tissues, based on a one way anova following the removal of non-changing genes. the number of differentially expressed genes identified suggested that expression varied significantly between tobacco tissues and throughout development for the majority of genes in the plant. tissues/organs in other plant species can be distinguished based on their transcriptional signature. principal components analysis was carried out on the tobea data to determine the relationship between tobacco tissues. figure shows the individual tobea microarrays plotted within the first three principal components. for each tissue type, the five biological replicate samples clustered closely together, suggesting that they were a reliable representation of the transcriptional profile for their respective tissue. the different tissue types could be broadly sub-divided into clusters based on their position within pc space. samples from imbibed seed showed distinct separation from the main body of samples along pc. the root and cotyledon/leaf samples were also separated along pc with the remaining tissue types falling between these groups. this suggested that pc was representative of some of the spatial variation in tobacco gene expression. some tissue types, such as roots, could not be distinguished based on the developmental stage, however, other tissue types did show a developmental separation along pcs and. this was apparent for the transition from floral apex to floral buds in cluster viii. greater separation in pc space for a developmental series was shown by leaves. cotyledon and young shoot samples along with young leaf samples showed clear separation from mature and senescing leaf samples along pcs and. this showed a clear distinction in transcriptional signature between young and old leaves. gene expression changes during tobacco leaf development to investigate the underlying cause of the separation between young and mature/senescing leaf material, genes were identified that showed differential expression across the'leaf' samples. genes were called differentially expressed if they showed significant variation and a minimum of five-fold change in expression for at least one pairwise comparison between young and mature leaf samples. the genes identified on this basis were clustered by k-means into groups as shown in figure two basic responses were represented by the clusters; genes showing relatively higher expression in younger samples and genes showing relatively higher expression in older samples. clusters were tested for over-representation of gene functions, based on the annotations borrowed from the best arabidopsis blast hit. when genes in clusters l and l were combined, the gene ontology analysis showed over-representation of multiple terms relating to photosynthesis and cell cycle, or cell growth and expansion. a combined list of the genes in clusters l and l showed over-representation of go terms relating to senescence related responses, including break down and re-mobilisation of various metabolites, hormone responses and stress responses. this suggested that there was a clear distinction between gene expression in young and old leaf material, with the former showing greater activity in photosynthesis, growth and expansion and the latter undergoing senescence. although the four clusters were defined by two basic responses, normalisation of the data across all of the tissues in tobea allowed further distinction of the clusters. for instance, genes in clusters l and l both showed relatively higher expression in younger leaves than older leaves, however, in the context of other tissues in the expression atlas, cluster l genes were relatively highly expressed in young leaf samples, whereas cluster l genes showed relatively low expression in older leaf samples. go analysis of the genes in cluster l principally showed terms associated with photosynthesis, whereas genes in cluster l showed terms related to cell cycle or cell growth and expansion. together, this suggested that the differences between young and old leaf tissues can be described as increased photosynthetic activity in younger samples and reduced cell division and growth along with increased senescence in older leaf tissues. this clearly shows the benefit of considering changes in gene expression in the context of all of the other tissue types in the tobea database and not just between a limited subset of tissue types. tissue specific tobacco gene expression the above analyses demonstrated that many tobacco genes show changes in expression between different tissues. to test for genes that are only expressed in specific tissues, all of the expressed genes were identified for each tissue type. for this analysis only genes flagged as present or marginal in all biological replicates of at least one developmental stage within a tissue type were considered expressed. figure shows a venn diagram for each of the six tissue types represented in the tobea database, with the genes identified as expressed in each tissue compared to a combined list of all those identified as expressed in the other tissues. go analysis was carried out on genes identified as expressed specifically in a single tissue type. the genes identified as specifically expressed in imbibed seed samples showed over-representation of terms associated with seed development and nutrient storage as well as those responsive to water and high light intensity, all processes/responses that are likely to be well represented in an imbibed seed. similarly, genes specifically expressed in root samples showed over-representation of terms related to nutrient uptake, transport and toxin or defence responses. those specifically expressed in leaf samples showed over-representation of genes involved in photosynthesis. additionally, genes specifically expressed in stem, shoot apices and flower buds/flowers showed over-representation of genes associated with microtubule development, implicated in the regulation of stem growth, chromosome segregation/dna metabolism and floral organ development respectively. co-expression of tobacco genes the set of genes showing differential expression across the tobea samples were analysed for co-expression. figure shows a subset of clusters from a set of k-means clusters. go analysis was carried out on the clusters and many of the identified biological processes could be related to the tissue types they showed expression in. for example genes in cluster k showed relatively high expression in imbibed seed samples and an over-representation of genes involved in seed development and nutrient storage. indeed 50% of the genes in cluster k were among the identified as specifically expressed in imbibed seed. similarly genes in cluster k showed relatively high expression in buds and flowers, and genes in clusters k showed relatively high expression in young leaves and these clusters showed over-representation of genes related to floral development and photosynthesis respectively. another example of expression of genes involved in relevant processes for the respective tissue is offered by cluster k these genes showed high expression in both root and stem samples and over-representation of genes involved in xylem and phloem development. clusters k13/k and k4/k8/k showed relatively high and relatively low levels of gene expression in mature and senescing leaf samples respectively. the former set of clusters showed over-representation of genes involved in senescence related processes and the latter for genes involved in cell cycle and growth. together with the results for cluster k this supported the suggestion that transcriptional signature differences between young and old leaf tissues was based on genes involved in photosynthesis, cell division and growth, or senescence. analysis of tobacco transcription factor expression patterns transcription factors play an important role in the regulation of gene expression. comprehensive surveys of the tfs present in several other plant species have been undertaken. although complete sequence coverage is not available for the tobacco genome, the gsr sequence data generated by the tgi allowed the identification of tfs in n. tabacum as part of the tobfac database. a blastn search was used to map these tfs to the tobacco unigenes assembled during this study. the tfs were mapped to unique unigenes, of which were represented by at least one probe set on the microarray. of the tobfac tfs mapped to probe sets on the array, 87% were identified as expressed in at least one tissue. figure shows the number and percentage of tobacco microarray probe sets representing tfs for each of the tobfac families scored as expressed. for the majority of the tf families over 60% of their members were scored as expressed. the exceptions to this were the shi related sequence and cpp families, where only 50% and 0% were expressed respectively. however, it should be noted that both these families were represented by two or less probe sets. the ethylene response factors are one of the largest tf families in tobacco with over members and are represented by probesets on the tobacco microarray. the erf family did not show any strong trend in tissue specificity within tobea, with a large number being expressed in all of the different tissue types. the erfs are divided into subgroups, and a high percentage of group ix tobacco erfs were previously shown to be induced in response to methyl jasmonate treatment. the group ix erfs were represented by probe sets on the tobacco microarray. compared to the family as a whole, the group ix erfs showed fewer members expressed in all other tissues aside from roots, where % of them were scored as expressed. meja treatment has been shown to inhibit root growth in arabidopsis and tobacco and it may be that the induction of these root expressed group ix erfs by meja plays an important role in the inhibition of growth. identification of putative transcriptional networks although expression could be measured for a majority of tfs across a range of different tissues, this does not mean that their expression levels were similar between tissues. correlation of expression level across different conditions provides a useful tool for functional genomics. the program biolayout 3d was used to generate gene networks based on co-expression of tfs across all of the arrays in tobea. individual probe sets for tfs are represented by nodes in the network and the edges between nodes represent correlated gene expression. of the probe sets representing tfs on the array, were placed into networks. figure shows an example of one of the networks generated including orthologs for mads-box tfs and one yabby tf involved in floral meristem identity in other plant species. it should be noted that an edge between two nodes does not necessarily indicate a causal interaction between these nodes as it could equally be interpreted as co-regulated expression of both nodes by a common regulator. however, several interactions suggested by the tobacco floral identity gene network were consistent with the gene regulatory network proposed for arabidopsis. for example putative interactions between apetala and ap between ap and agamous and both of these genes with pistillata in tobacco are supported by results in arabidopsis. some interactions shown in arabidopsis were not represented in the tobacco tf network, such as a positive interaction between ap and pi and a negative interaction between ag and ap failure to see the latter interaction is not surprising as the network is based on positive correlation. the tf network also suggests a positive interaction between ap and agamous like, whereas agl is reportedly repressed by ap in arabidopsis. this suggests a different relationship may exist between these genes in tobacco compared to arabidopsis. two putative tobacco agl orthologs were identified, so it was possible that only one had lost the repression by ap however, the other agl ortholog was placed in a separate small network along with another ap ortholog, suggesting that both of the tobacco agl orthologs were not repressed by ap. the tobacco tf network appeared to consist of two distinct sub-clusters, one including agl and ap orthologs and the other including orthologs for ap pi, ag, crabs claw, sepallata and seedstick. this network structure is consistent with the former two genes being involved earlier on during floral determination, whereas the remaining genes have been implicated later in floral development and floral organ identity. development of web-based tools for tobea the tobacco microarray was designed from a unigene assembly based on sequences. since the assembly was carried out there has been a significant increase in the sequence data available for tobacco, with over k ests currently available at genbank. a more recent set of unigenes was assembled from n. tabacum sequences by the sgn. the sgn also houses nicotianacyc, a database linking the tobacco unigenes to the metacyc biochemical pathways. to benefit from the greater genomic coverage and annotation information, unigenes present on the microarray were mapped to the current sgn unigene assembly. of the probe sets on the microarray could be mapped to of the sgn unigenes, indicating approximately 38% coverage of the sgn unigene build by the microarray. approximately 81% of the mapped sgn unigenes were represented by a unique probe set, and over 99% by probe sets or less. information of the temporal and spatial expression pattern of genes, along with co-expression across a broad range of conditions can be very useful in inferring function for unknown genes. in order to make the information contained within tobea more accessible to other researchers and enable them to place information on their gene of interest within the context of its expression information, it was decided to integrate the tobea expression data into the sgn website. figure shows some sample screen shots of the website, where users can access expression information on their gene of interest. the sgn expression data module allows users to view the expression level of their unigene across the different samples included in tobea. the sedm also allows users to identify other sgn unigenes that show correlated expression across the tobea microarray data set. discussion recent efforts, such as the tgi, have massively increased the sequence resources available for n. tabacum. however, the ests generated by this study still represent a significant contribution to the total number of ests available for tobacco and will prove very useful in applications such as the detection of varietal polymorphisms or gene model predictions. along with other available sequences, these ests were assembled into a set of unigenes and used to design a custom affymetrix expression microarray for tobacco. affymetrix chips are a proven and widely accepted technology and the design service has been used successfully to generate a large number of custom chips for other research applications http://www.affymetrix.com. the resultant microarray was used to capture the temporal and spatial changes in gene expression throughout the lifecycle of the tobacco plant. considerable variation in gene expression was shown across the different tissues within tobea, with the transcriptional signatures revealing relationships between samples. as observed for arabidopsis the transcriptional profile of imbibed seed showed the greatest difference to all other samples causing them to be distinctly removed in principal component space. pca also showed a broad distinction between samples originating from root and shoot tissues. studies in other plants have shown that the complement of genes expressed in a tissue is related to the biological function that tissue performs. analysis of the go annotations shows that this is also true in tobacco. as well as spatial changes, temporal changes in gene expression were apparent over developmental series in the same tissues. this was more apparent for some tissues than others. for example pca showed little separation between young and mature roots. there was, however, a tendency for stronger expression of genes related to cell division, growth and expansion in younger tissues and stronger expression of senescence related genes in older tissues. on this basis there was a clear separation between young and old leaf samples, consistent with findings for arabidopsis. consistency of the results contained within tobea with previous results in other plants suggests that it is a high quality expression data set and a good representation of the spatial and temporal changes in gene expression throughout the lifecycle of the tobacco plant. together with close clustering of independent replicate samples this also indicates that the microarray can be used to reliably and reproducibly measure gene expression in tobacco. since the study was initiated larger unigene sets have been assembled for tobacco from the increased amount of sequence data available. these new unigene assemblies likely represent greater coverage of the tobacco genome. mapping of the microarray probe sets to the current unigene build from the sgn showed that the microarray still represents around 38% coverage of tobacco genes. mapping of the probe sets to any future unigene assemblies will be maintained to ensure that the annotation information available for the array remains up to date. as demonstrated by tobfac, the tobacco gsr sequence data provides the potential to identify genes that are not represented in the available est libraries. this is most likely the case for rare transcripts or those showing temporal, spatial, or alternative forms of regulation that is not captured in the conditions sampled by the current est libraries. in future assemblies, the incorporation of sequence data such as gsrs with est sequences has the potential to provide more comprehensive coverage of the genes present in the tobacco genome than the est data alone. the microarray represents a very valuable tool for research in tobacco, allowing comprehensive expression studies to be carried out in the plant. to maximise the benefit of this the array has been made publicly available for use via the affymetrix service at nasc http://affymetrix.arabidopsis.info. the microarray might also provide a very useful tool to researchers in closely related species, in particular other nicotiana species, via cross-species hybridisations. tobea provides a useful reference data set to any future experiments carried out using the microarray, allowing a more detailed characterisation of the differentially expressed genes identified. to facilitate use of the information contained in tobea, a set of web-based tools were developed. these tools currently allow researchers to view the expression level of their gene of interest across the different tissues in the atlas and identify other genes that show highly correlated expression via the sgn website. this will enable individual users to carry out functional genomic analysis allowing the annotation of the sgn unigenes to be updated by experts from all different fields of biology via the locus editor feature of the sgn website. as further experiments are carried out using the tobacco microarray, the data will be uploaded into the sgn expression database sedm. the sedm can store results for any solanaceae species, and is capable of storing data sets from both microarray and direct sequencing studies. enabling users to upload expression data to the sgn expression database means it has the potential to become a very useful resource for all researchers in the solenaceae. the value of tobea is demonstrated by the study of specific sets of genes such as transcription factors. due to their regulatory role, tfs represent good targets for gross modifications of biochemical or signalling pathways for various biotechnological applications. over 30% of the tfs identified by rushton et al., in a genome scale survey of tobacco could be mapped to unigenes on the microarray. simple correlation of tf expression levels across the tissues in tobea enabled the construction of a gene network involved in floral determination and floral organ identity. several of the connections observed in this network were consistent with known interactions from other plant species. extending this type of network analysis to include genes beyond tfs could allow the identification of putative transcriptional networks for specific metabolic or signalling pathways. this analysis could identify regulatory targets to enable the manipulation of these pathways in efforts to increase or decrease the production of various secondary metabolites in tobacco.  an affymetrix microarray was developed that allows reliable and reproducible measurement of gene expression in tobacco. this array is available for public use via the nasc affymetrix service. the array was used to produce the tobacco expression atlas, which provides a snap-shot of gene expression from multiple tissues throughout the lifecycle of the tobacco plant. a set of web based tools were developed in order to facilitate public access to this data via the sgn web site http://solgenomics.net/. it is intended that the database will be extended in future with the addition of further expression datasets, not only from tobacco but other plants of the solanaceae family. the sgn welcomes the donation of such data from the research community. tobea represents a valuable resource for functional genomic analysis in plants and has potential application in research leading to biotechnological and agronomic improvements to tobacco as well as related species. 
tobea provides a snap shot of the transcriptional activity for thousands of tobacco genes in different tissues throughout the lifecycle of the plant and enables the identification of the biological processes occurring in these different tissues. a custom designed affymetrix tobacco expression microarray was generated from a set of over 40k unigenes and used to measure gene expression in different tobacco samples to produce the tobacco expression atlas. transcriptomics has resulted in the development of large data sets and tools for the progression of functional genomics and systems biology in many model organisms. the data provided by tobea represents a valuable resource for plant functional genomics and systems biology research and can be used to identify gene targets for both fundamental and applied scientific applications in tobacco. the array has also been made available for public use via the nottingham arabidopsis stock centre microarray service. several interactions in a floral identity transcription factor network were consistent with previous results from other plant species. putative transcriptional networks were identified based on the co-expression of these transcription factors. currently there is no commercially available microarray to allow such expression studies in nicotiana tabacum. of transcription factors previously identified in tobacco were mapped to the array, with 87% of them being expressed in at least one tissue in the atlas. to broaden access and maximise the benefit of tobea a set of tools were developed to provide researchers with expression information on their genes of interest via the solanaceae genomics network web site.
 transcriptomics has resulted in the development of large data sets and tools for the progression of functional genomics and systems biology in many model organisms. currently there is no commercially available microarray to allow such expression studies in nicotiana tabacum.  a custom designed affymetrix tobacco expression microarray was generated from a set of over 40k unigenes and used to measure gene expression in different tobacco samples to produce the tobacco expression atlas. tobea provides a snap shot of the transcriptional activity for thousands of tobacco genes in different tissues throughout the lifecycle of the plant and enables the identification of the biological processes occurring in these different tissues. of transcription factors previously identified in tobacco were mapped to the array, with 87% of them being expressed in at least one tissue in the atlas. putative transcriptional networks were identified based on the co-expression of these transcription factors. several interactions in a floral identity transcription factor network were consistent with previous results from other plant species. to broaden access and maximise the benefit of tobea a set of tools were developed to provide researchers with expression information on their genes of interest via the solanaceae genomics network web site. the array has also been made available for public use via the nottingham arabidopsis stock centre microarray service.  the generation of a tobacco expression microarray is an important development for research in this model plant. the data provided by tobea represents a valuable resource for plant functional genomics and systems biology research and can be used to identify gene targets for both fundamental and applied scientific applications in tobacco. 