BACKGROUND
l-ascorbic acid  is essential for most of the living plant tissues.
in addition to its known function as an antioxidant, asa has important roles in plant cell growth and expansion, photosynthesis, and hormonal regulation  <cit> .
humans are unable to synthesize asa due to mutations in the enzyme l-gulono- <dig> -lactone oxidase  that catalyzes the final step of its biosynthesis, and as consequence need to incorporate asa through the dietary consumption of fresh fruits and vegetables .
although asa deficiency is not a current problem in developed countries, it is recognized that high asa dietary consumption has important health benefits for the consumer, and an increased intake of asa has been associated with a decreased incidence of several important human diseases and disorders  <cit> .
so far, the study of genes regulating asa content in plants has been tackled mainly through the use of reverse genetics targeting asa biosynthesis and recycling genes  <cit> .
even though classical genetic approaches has resulted in the cloning of genes responsible for complex traits  <cit> , and several qtls have been identified for asa, yet the genes underlying these qtls need to be determined  <cit> .
this is likely because identification of the gene responsible for a qtl is time-consuming and technically demanding especially when the trait requires careful analytical techniques as it is the case for asa.
in order to overcome these drawbacks, the use of omics approaches to study complex traits have emerged during the past years, and the use of global transcript abundance together with gene cluster analysis has become a useful approach to predict and in some cases assign gene functions  <cit> .
the premise behind clustering analysis is that genes having similar expression profiles across a set of conditions  may share similar functions or be involved in similar processes  <cit> .
still, it is clear that genes with the similar functions do not necessarily share similar transcriptional patterns, and conversely.
despite this, it has been shown that large numbers of functionally related genes show very similar expression patterns under a relevant set of conditions, especially genes that are co-regulated by common transcription factors, or whose products are components of multiprotein complexes  <cit> .
for these reasons, clustering genes with similar expression patterns may allow to assign putative functions to unknown genes via “guilt-by-association”  <cit> .
in order to accomplish this, several clustering techniques such as hierarchical clustering   <cit> , self-organizing map   <cit> , and self-organizing tree algorithm   <cit>  have been successfully used in tomato to identify genes with correlated expression that turned out in the selection of candidate genes for further functional analyses.
as an example, the transcriptomic comparison of a s. pennellii introgression line  with its recurrent s. lycopersicum parental line, identified a pectinesterase and two polygalacturonase genes that were associated to the different asa accumulation in the il fruits  <cit> .
in this study, several genes that correlate with asa content in tomato fruit have been identified through the use of an algorithm designed to draw functional associations among differentially expressed genes.
these possible associations are discussed in terms of the physiological processes that might ultimately regulate the asa content in tomato fruits, and by mining arabidopsis expression data repositories we identify cues that may be triggering expression changes similar to those associated with the asa content in red tomato fruits.
RESULTS
identification of tomato rils with contrasting content of asa in ripe fruits
a population of  <dig> recombinant inbred lines   generated from an inter-specific cross of the domesticated species solanum lycopersicum cv.
moneymaker  and the wild species solanum pimpinellifollium  has been evaluated for asa content in two consecutive years .
pericarp tissue from at least three red ripe fruits per plant and three plants per genotype were evaluated for asa content using high performance liquid chromatography .
figure  <dig> shows the distribution of asa contents in the ril population when expressed as fresh weight  and dry weight  for two years harvests.
as expected, a continuous asa content distribution was observed due to the polygenic nature of asa accumulation  <cit> .
table  <dig> shows the asa content in the parental and the range in the rils over the two years evaluated.
range in the ril population progeny expressed as fresh  and dry weight .
similar to previous reports on another tomato wild relative s. pennelli <cit> , the asa content of s. pimpinellifollium was higher than that of the cultivated tomato s. lycopersicum mm by about 2-fold on a fresh weight basis , and increased to around 6-fold when expressed as dry weight  .
the asa content was lower in both parents in the year  <dig> and the same was true for the rils when expressed on a fw basis indicating an important environmental influence in this trait .
thus, asa content ranged from  <dig>  mg/100 g fw to  <dig>  mg/100 g fw in the year  <dig> and from  <dig>  mg/100 g fw to  <dig>  mg/100 g fw in the year  <dig> .
asa content in the population remained significantly -although weakly- stable as assessed for repeatability by the correlation coefficient .
however, when results were expressed relative to dw, the range of asa remained approximately the same but the asa content in most of the rils were not consistent during both years .
figure  <dig> depicts the ten rils lines with the most extreme values of asa based on the asa/fw values consistently obtained for the two years.
a two-way anova with data from the ten rils selected and the parents ‘moneymaker’ mm and to- <dig>  using “genotype” and “year” as factors, reported significant effect of genotype  and year  and no significant interaction between them  .
the genotype explained almost 83% of the variance  validating the selection of these ten rils for further analysis .
*all f-ratios are based on the residual mean square error.
- type iii sums of squares.
identification of genes whose expression correlated with asa content in tomato fruits
next, we aimed to identify tomato genes whose expression correlated with the asa content in red ripe fruits from the ten rils previously selected.
for that purpose, differential transcript abundance was analyzed using two-color hybridizations and the tom <dig> array , in which according to the unigene set # <dig> of the sol genomic network   <dig>  tomato sequences are represented  <cit> .
probes were obtained from: the five individual rils that contained high asa, the five individual rils that contained low asa, plus two probes generated with a mix of the five of each set.
three biological replicates were hybridized and normalized using as a common reference a sample containing a bulk rna including all high and low asa content rils, and dye-swap was performed.
our analysis resulted in the identification of a number of genes whose expression profile correlated with the high or low asa content in the  <dig> rils .
using a strict criteria in the significant analysis of microarray  we identified  <dig> genes with at least 2-fold change in expression between rils having high and low asa content .
forty-four positivevitaminc co-regulated  genes  and  <dig> negativevitaminc co-regulated  genes  were identified .
in order to validate the microarray results, qrt-pcrs were performed in one of each group of two contrasting rils for several genes showing differential expression in the microarray .
the qrt-pcr expression of the selected genes was in accordance with microarray data and followed the same trends in the individual lines analyzed.
functional classification of the pvc and nvc genes
the  <dig> differentially expressed genes were classified by functional categories according to the go slim tool available in the tomato expression database .
the functional categorization of the  <dig> pvc genes showed that  <dig> genes were classified as nucleotide binding,  <dig> genes encoded proteins showing homology to kinases, and  <dig> genes encoded putative transporters.
among the  <dig> nvc genes,  <dig> were classified as transcription factors,  <dig> were classified as kinases, and one as a transporter .
the tomato tom <dig> microarray contains oligonucleotides against  <dig> genes encoding for enzymes of the wheeler-smirnoff pathway, the main asa biosynthetic pathway in tomato  <cit>  and for all  <dig> genes involved in asa recycling.
our results fail to identify among the differentially expressed pvc or nvc candidates any of the above-mentioned genes indicating a lack of co-regulation between the asa metabolic pathway and the asa content in red tomato fruits from our ril population .
this result is in accordance with a previous study showing that asa biosynthetic genes are not the main regulators of asa content in red tomato fruit  <cit> .
however, we cannot discard that genes involved in asa biosynthesis or recycling determine the asa accumulation during the fruit ripening.
nevertheless we paid special attention to biosynthetic genes whose expression has been suggested to control asa content during ripening such as gpp <dig> <cit> , ggp <dig> and ggp <dig> <cit> .
however we did not find any differential expression of these genes that might account for the reported content in asa among the rils, indicating that their expression do not determine the final asa content in tomato fruit in the genetic background of our rils population.
mdhr <dig>  involved in asa recycling, has been previously reported as a likely candidate for being qtl for asa content in tomato fruit in studies with the s. pennellii il population  <cit> .
however, it was not identified as differentially expressed in the ril population here analyzed.
therefore, whether the genes regulating asa content are species-dependent or the result of polymorphisms eventually resulting in differences in enzymatic activity deserves further investigation.
based on our results we hypothesize that the genetic factors determining the final asa content in ripe tomato fruits in our ril population could be the result of altered expression of genes other than asa biosynthesis, and the analysis of the functional categories classification from the pvc and nvc genes should help us to identify those genes.
clustering of co-regulated genes using a self-organizing tree algorithm sota
in order to identify relationships among differentially expressed genes and the asa content in tomato we performed a self-organizing tree algorithm   <cit>  using pearson as metrics.
these analyses grouped the transcript abundance profiles in  <dig> clusters:  <dig> clusters grouped the  <dig> pvc genes  and  <dig> clusters grouped the  <dig> nvc genes .
the identity of the genes belonging to each cluster is shown in table  <dig>  which also includes the unigene annotation based on the sol genomic networks web site , and the corresponding arabidopsis ortholog identified using blast2go  <cit> .
next, we analyzed in more detail the identity and relationships among members belonging to individual pvc and nvc clusters.
unigene annotations based on the sol genomic networks web site , and the accession numbers corresponding to arabidopsis orthologs identified using blast2go  <cit>  are shown for those genes whose expression is depicted in figure  <dig>
among the genes found in cluster  <dig> there were two with homology to abi genes , a guanylate kinase, and an ethylene responsive chitinase b <cit>  .
abi genes encode protein phosphatases 2 c  that are negative regulators of aba responses through their binding and dephosphorylation of snrk <dig>  a core component of the cytoplasmic aba receptors  <cit> .
the connection between aba and asa has been previously established because concomitant upregulation of abi <dig> and enzymes from the ascorbate-glutathione cycle has been recently reported in aba-treated a. thaliana <cit> .
cluster  <dig> contains two auxin-regulated proteins, one of them, the aux/iaa transcription factor slaa <dig>  shows an ethylene ripening-associated expression pattern  <cit> , and its transcript accumulation is dramatically reduced in the tomato ripening mutants rinnor, and nr <cit> .
the rinnor, and nr mutants lack the capacity to respond to the autocatalytic production of ethylene and to further undergo normal ethylene-regulated ripening processes  <cit> .
therefore, it has been proposed that, ethylene modulates auxin responsiveness during tomato fruit ripening through slaa <dig>  <cit> .
the second auxin regulated protein is the xyloglucan endotransglucosylase-hydrolase- <dig>  gene which is expressed during fruit ripening and its expression peaks at the red stage concomitantly with the maximum of ethylene production  <cit> .
the second auxin regulated protein is the xyloglucan endotransglucosylase-hydrolase- <dig>  gene which is expressed during fruit ripening and its expression peaks at the red stage concomitantly with the maximum of ethylene production  <cit> .
clusters  <dig> and  <dig> were closely related and grouped elements that likely have a direct role in the accumulation of asa such as an ascorbate peroxidase, a putative chloroplastic glutathione reductase, two glutathione-s-transferases , and several kinases.
the up-regulation of a putative glutathione reductase together with the consumption of asa  suggests that the high asa rils could also have higher asa recycling rates.
these results contrast with a previous study reporting that a putative ascorbate peroxidase and a putative glutathione-s-transferase genes were down-regulated in an inbred line selected for its high asa content when compared with the parental line  <cit> .
the different results derived from both studies suggest that asa recycling rate and asa content might be uncoupled events in the tomato rils here employed.
the  <dig> genes down-regulated in the rils group with the highest content of asa could be grouped in  <dig> clusters .
cluster  <dig> includes the s. lycopersicum 1-aminocyclopropane-1-carboxylate  oxidase gene , whose product is an enzyme involved in ethylene biosynthesis that uses asa as a cofactor.
the silencing of the acc oxidase gene in tomato is responsible for up to 87% reductions of ethylene in ripening fruits  <cit> .
the acc oxidase down-regulation together with the concomitant down-regulation of a bhlh transcription factor ethylene responsive  <dig>  found in the same cluster seems to point to a reduced ethylene production in the rils with higher asa content.
this is further supported by the down-regulation of a gene coding for a s. lycopersicum squamosa promoter binding-like  protein in cluster  <dig>  and the s. lycopersicum green ripe-like  <dig>  in cluster  <dig>  spbl is the gene affected in the tomato colorless nonripening  mutant that results in colorless fruits with a substantial loss of cell-to-cell adhesion  <cit> .
grl <dig> encodes for the closest homolog in tomato of the green ripe gene responsible for the non-ripening phenotype found in the dominant green-ripe  tomato mutant  <cit> .
both, spbl and grl <dig> are thought to be involved in ethylene responses during tomato fruit ripening, being major regulatory elements in the network controlling the process.
in order to discard possible differences in the ripening stage between the two groups of contrasting asa genes that could account for the differential expression of ethylene-related genes we searched for their expression pattern during ripening in the tomato expression database  or in the literature.
for instance, according to ted, the expression of s. lycopersicum 1-aminocyclopropane-1-carboxylate  oxidase gene  does not vary significantly between breaker and red stage.
on the other hand, it has been reported that the expression of er <dig>  is induced in red stage and does not vary from mature green to turning  <cit> , so if the differential expression were due to differences in the ripening stage, they should have occurred in all rils from one group, something highly unlikely.
these data allowed us to discard differences in ripening stages as the source of differential expression in ethylene-related genes.
clusters  <dig> and  <dig> include different elements involved in the responses to gibberellins, such as the s. lycopersicum gibberellin 20-oxidase- <dig> gene and the putative gid <dig> f-box gene.
the gibberellin 20-oxidase- <dig> is one of the three ga 20-oxidases  involved in the 13-hydroxilation gibberellin biosynthesis pathway.
in this pathway, the intermediate ga <dig> is converted to ga <dig>  to be subsequently hydroxilated to the bioactive ga <dig> form by ga 3-ox  <cit> .
ga 20-oxidases are main regulatory control points of the gibberellin biosynthesis pathway  <cit> .
the rice gid <dig> f-box protein is part of the scf -type e <dig> ubiquitin ligase scfgid2/sly <dig> that positively regulates gibberellin signaling through the degradation of specific della repressors  <cit> .
the differential regulation of genes implicated in gibberellin biosynthesis, with positive correlation of ga- <dig> ox in cluster  <dig> and negative correlation of ga- <dig> ox in cluster  <dig> is remarkable, opening the possibility that ga influences, points to a non-previously described interaction between gibberellin and asa content in tomato.
cluster  <dig> contains a gene encoding for a putative udp-sugar transporter  .
how asa transport occurs between compartments of the cell and the apoplast remains largely unknown, as well as the transport of the several intermediates for its synthesis  <cit> .
in many cases, ndp-sugars involved in asa biosynthesis, such as udp-glucose, udp-glucuronic acid and gdp-mannose, are available in the cytosol, and likely require specific transporters to pass through the endomembrane system for further processing.
the dow-nregulation of an udp-sugar transporter in the rils containing more asa might be indicative of the importance of ndp-sugar transporters in the final content of asa in a plant tissue.
cluster  <dig> groups two cell wall related genes, a putative cellulose synthase and a xyloglucan endotransglycosylase precursor slxet <dig> induced by brassinoesteroids  <cit> .
other cell wall modifying enzyme, the s. lycopersicum endoxyloglucan transferase , was identified in cluster  <dig>  this result might reveal a link between cell wall modification, degradation, and asa content  <cit> .
normally, slxet <dig> appears strongly down-regulated at the beginning of turning stage of fruit ripening and appears undetectable at the red stage  <cit> , in contrast to slxet <dig>  whose expression drops at the turning and pink stages, to raise again at the red stage.
finally, a putative cytochrome p <dig> whose expression pattern resembles that of slxet <dig> was also identified in cluster  <dig>  cytochromes p <dig>  many of which are involved in the biosynthesis and metabolism of brassinosteroids  <cit> , are known to control the redox status of the cell  <cit> .
the fact that several elements controlling the plant redox status were identified in our analysis lead us to hypothesize that this process might be determinant for the asa accumulation in tomato fruit .
genes correlated with asa content in tomato fruit are regulated by oxidative stress
databases for co-expression analysis have been established for the model plant arabidopsis thaliana, which can be used to get insights into the mechanisms underlying the differential transcript abundance in our contrasting asa lines.
as result, various repositories of transcriptome data are now available including nascarrays,  <cit>  geo,  <cit>  smd,  <cit>  arrayexpress,  <cit>  and atgenexpress,  <cit> , which collectively provide  <dig>  microarray data points .
using this repository, we have searched for the most homologous pvc and nvc genes in arabidopsis , and we have analyzed using a correlation analysis of the clustering tools in genevestigator v <dig>  <cit>  whether different experimental conditions were reported to cause the transcriptional induction or repression of these sets of genes.
the results of our co-expression datasets were analyzed independently for pvc and nvc genes using this huge data set repository.
we were expecting that if the pvc and nvc set of genes have a collective biological significance they must be regulated by similar conditions in an opposite way.
we identified that  <dig> % of the atpvc genes were induced by flagellin <dig>  .
importantly, we found that  <dig> % of atnvc genes were repressed by flag <dig> treatment in an independent correlation analysis .
the fact that independent correlation analyses using atpvc and atnvc genes rendered similar treatments in arabidopsis clearly indicates that these are relevant biological associations.
early events caused by flag <dig> treatments include ion fluxes across the plasma membrane and the formation of reactive oxygen species  <cit> .
this relationship of pvc and nvc genes with a ros-generating agent adds further support to the association of asa content and the maintenance of the redox state, as deduced from the clustering analysis.
CONCLUSIONS
the differentially expressed genes that correlate with asa content in tomato fruits identified in the present study suggest a scenario where several genes involved in hormone responses are interlinked to modulate the content of asa .
cross-talk among different hormones has been reported to regulate several processes in plants: gibberellins and auxin in tomato fruit set  <cit> ; gibberellins and aba during seed germination  <cit> ; gibberellins and ethylene, in root elongation  <cit> ; aba and ethylene, in the modulation the overall carbon status during early seedling growth and development  <cit> ; brassinosteroids and auxin in the control of lateral root growth  <cit> , etc.
ethylene has been implicated indirectly in the regulation of reduced asa content in pericarp prior to tomato ripening as the ethylene-insensitive nr mutant accumulates almost 2-fold asa compared to wild type during mature green and breaker stages  <cit> .
aba has also been connected with the increase of asa content through promoting its recycling  <cit> .
the co-regulation analyses in arabidopsis using the pvc and nvc orthologs identified that these genes are similarly regulated by flag <dig>  whose primary effect is the generation of ros in response to pathogen attack  <cit> .
these results suggest that asa content in tomato fruit might be critically regulated by hormone interactions, and it is directly related to the oxidative status of the fruit, as previously suggested  <cit> .
however an important aspect that needs to be considered is the likely possibility that part of the asa in the tomato fruit as has been recently reported.
therefore part of the fruit asa, in addition of in situ biosynthesis, is likely to be provided by source organs complicating even more the identification of asa determinants  <cit> .
current approaches have been unfruitful in explaining the natural variability of asa content in tomato fruit.
the combination of different omics approaches, surveying several metabolites, genes, and gene products seems to be the most realistic way of finding out the source of complex traits like asa content, whose variation among different tomato species is intricately related with hormones  <cit> .
the exhaustive analyses of the omics data provides a more rational picture of the actual physiological processes determining the asa fruit content, and would allow to target sets of co-expressed genes to be further studied by functional analyses.

 l-ascorbic acid is essential for most of the living plant tissues. in addition to its known function as an antioxidant, asa has important roles in plant cell growth and expansion, photosynthesis, and hormonal regulation. humans are unable to synthesize asa due to mutations in the enzyme l-gulono- -lactone oxidase that catalyzes the final step of its biosynthesis, and as consequence need to incorporate asa through the dietary consumption of fresh fruits and vegetables. although asa deficiency is not a current problem in developed countries, it is recognized that high asa dietary consumption has important health benefits for the consumer, and an increased intake of asa has been associated with a decreased incidence of several important human diseases and disorders. so far, the study of genes regulating asa content in plants has been tackled mainly through the use of reverse genetics targeting asa biosynthesis and recycling genes. even though classical genetic approaches has resulted in the cloning of genes responsible for complex traits, and several qtls have been identified for asa, yet the genes underlying these qtls need to be determined. this is likely because identification of the gene responsible for a qtl is time-consuming and technically demanding especially when the trait requires careful analytical techniques as it is the case for asa. in order to overcome these drawbacks, the use of omics approaches to study complex traits have emerged during the past years, and the use of global transcript abundance together with gene cluster analysis has become a useful approach to predict and in some cases assign gene functions. the premise behind clustering analysis is that genes having similar expression profiles across a set of conditions may share similar functions or be involved in similar processes. still, it is clear that genes with the similar functions do not necessarily share similar transcriptional patterns, and conversely. despite this, it has been shown that large numbers of functionally related genes show very similar expression patterns under a relevant set of conditions, especially genes that are co-regulated by common transcription factors, or whose products are components of multiprotein complexes. for these reasons, clustering genes with similar expression patterns may allow to assign putative functions to unknown genes via “guilt-by-association”. in order to accomplish this, several clustering techniques such as hierarchical clustering, self-organizing map, and self-organizing tree algorithm have been successfully used in tomato to identify genes with correlated expression that turned out in the selection of candidate genes for further functional analyses. as an example, the transcriptomic comparison of a s. pennellii introgression line with its recurrent s. lycopersicum parental line, identified a pectinesterase and two polygalacturonase genes that were associated to the different asa accumulation in the il fruits. in this study, several genes that correlate with asa content in tomato fruit have been identified through the use of an algorithm designed to draw functional associations among differentially expressed genes. these possible associations are discussed in terms of the physiological processes that might ultimately regulate the asa content in tomato fruits, and by mining arabidopsis expression data repositories we identify cues that may be triggering expression changes similar to those associated with the asa content in red tomato fruits.  identification of tomato rils with contrasting content of asa in ripe fruits a population of recombinant inbred lines generated from an inter-specific cross of the domesticated species solanum lycopersicum cv. moneymaker and the wild species solanum pimpinellifollium has been evaluated for asa content in two consecutive years. pericarp tissue from at least three red ripe fruits per plant and three plants per genotype were evaluated for asa content using high performance liquid chromatography. figure  shows the distribution of asa contents in the ril population when expressed as fresh weight and dry weight for two years harvests. as expected, a continuous asa content distribution was observed due to the polygenic nature of asa accumulation. table  shows the asa content in the parental and the range in the rils over the two years evaluated. range in the ril population progeny expressed as fresh and dry weight. similar to previous reports on another tomato wild relative s. pennelli, the asa content of s. pimpinellifollium was higher than that of the cultivated tomato s. lycopersicum mm by about 2-fold on a fresh weight basis, and increased to around 6-fold when expressed as dry weight. the asa content was lower in both parents in the year and the same was true for the rils when expressed on a fw basis indicating an important environmental influence in this trait. thus, asa content ranged from  mg/100 g fw to  mg/100 g fw in the year and from  mg/100 g fw to  mg/100 g fw in the year. asa content in the population remained significantly -although weakly- stable as assessed for repeatability by the correlation coefficient. however, when results were expressed relative to dw, the range of asa remained approximately the same but the asa content in most of the rils were not consistent during both years. figure  depicts the ten rils lines with the most extreme values of asa based on the asa/fw values consistently obtained for the two years. a two-way anova with data from the ten rils selected and the parents ‘moneymaker’ mm and to- using “genotype” and “year” as factors, reported significant effect of genotype and year and no significant interaction between them. the genotype explained almost 83% of the variance validating the selection of these ten rils for further analysis. *all f-ratios are based on the residual mean square error. - type iii sums of squares. identification of genes whose expression correlated with asa content in tomato fruits next, we aimed to identify tomato genes whose expression correlated with the asa content in red ripe fruits from the ten rils previously selected. for that purpose, differential transcript abundance was analyzed using two-color hybridizations and the tom array, in which according to the unigene set # of the sol genomic network tomato sequences are represented. probes were obtained from: the five individual rils that contained high asa, the five individual rils that contained low asa, plus two probes generated with a mix of the five of each set. three biological replicates were hybridized and normalized using as a common reference a sample containing a bulk rna including all high and low asa content rils, and dye-swap was performed. our analysis resulted in the identification of a number of genes whose expression profile correlated with the high or low asa content in the rils. using a strict criteria in the significant analysis of microarray we identified genes with at least 2-fold change in expression between rils having high and low asa content. forty-four positivevitaminc co-regulated genes and negativevitaminc co-regulated genes were identified. in order to validate the microarray results, qrt-pcrs were performed in one of each group of two contrasting rils for several genes showing differential expression in the microarray. the qrt-pcr expression of the selected genes was in accordance with microarray data and followed the same trends in the individual lines analyzed. functional classification of the pvc and nvc genes the differentially expressed genes were classified by functional categories according to the go slim tool available in the tomato expression database. the functional categorization of the pvc genes showed that genes were classified as nucleotide binding, genes encoded proteins showing homology to kinases, and genes encoded putative transporters. among the nvc genes, were classified as transcription factors, were classified as kinases, and one as a transporter. the tomato tom microarray contains oligonucleotides against genes encoding for enzymes of the wheeler-smirnoff pathway, the main asa biosynthetic pathway in tomato and for all genes involved in asa recycling. our results fail to identify among the differentially expressed pvc or nvc candidates any of the above-mentioned genes indicating a lack of co-regulation between the asa metabolic pathway and the asa content in red tomato fruits from our ril population. this result is in accordance with a previous study showing that asa biosynthetic genes are not the main regulators of asa content in red tomato fruit. however, we cannot discard that genes involved in asa biosynthesis or recycling determine the asa accumulation during the fruit ripening. nevertheless we paid special attention to biosynthetic genes whose expression has been suggested to control asa content during ripening such as gpp, ggp and ggp. however we did not find any differential expression of these genes that might account for the reported content in asa among the rils, indicating that their expression do not determine the final asa content in tomato fruit in the genetic background of our rils population. mdhr involved in asa recycling, has been previously reported as a likely candidate for being qtl for asa content in tomato fruit in studies with the s. pennellii il population. however, it was not identified as differentially expressed in the ril population here analyzed. therefore, whether the genes regulating asa content are species-dependent or the result of polymorphisms eventually resulting in differences in enzymatic activity deserves further investigation. based on our results we hypothesize that the genetic factors determining the final asa content in ripe tomato fruits in our ril population could be the result of altered expression of genes other than asa biosynthesis, and the analysis of the functional categories classification from the pvc and nvc genes should help us to identify those genes. clustering of co-regulated genes using a self-organizing tree algorithm sota in order to identify relationships among differentially expressed genes and the asa content in tomato we performed a self-organizing tree algorithm using pearson as metrics. these analyses grouped the transcript abundance profiles in clusters: clusters grouped the pvc genes and clusters grouped the nvc genes. the identity of the genes belonging to each cluster is shown in table  which also includes the unigene annotation based on the sol genomic networks web site, and the corresponding arabidopsis ortholog identified using blast2go. next, we analyzed in more detail the identity and relationships among members belonging to individual pvc and nvc clusters. unigene annotations based on the sol genomic networks web site, and the accession numbers corresponding to arabidopsis orthologs identified using blast2go are shown for those genes whose expression is depicted in figure  among the genes found in cluster there were two with homology to abi genes, a guanylate kinase, and an ethylene responsive chitinase b. abi genes encode protein phosphatases 2 c that are negative regulators of aba responses through their binding and dephosphorylation of snrk a core component of the cytoplasmic aba receptors. the connection between aba and asa has been previously established because concomitant upregulation of abi and enzymes from the ascorbate-glutathione cycle has been recently reported in aba-treated a. thaliana. cluster contains two auxin-regulated proteins, one of them, the aux/iaa transcription factor slaa shows an ethylene ripening-associated expression pattern, and its transcript accumulation is dramatically reduced in the tomato ripening mutants rinnor, and nr. the rinnor, and nr mutants lack the capacity to respond to the autocatalytic production of ethylene and to further undergo normal ethylene-regulated ripening processes. therefore, it has been proposed that, ethylene modulates auxin responsiveness during tomato fruit ripening through slaa. the second auxin regulated protein is the xyloglucan endotransglucosylase-hydrolase- gene which is expressed during fruit ripening and its expression peaks at the red stage concomitantly with the maximum of ethylene production. the second auxin regulated protein is the xyloglucan endotransglucosylase-hydrolase- gene which is expressed during fruit ripening and its expression peaks at the red stage concomitantly with the maximum of ethylene production. clusters and were closely related and grouped elements that likely have a direct role in the accumulation of asa such as an ascorbate peroxidase, a putative chloroplastic glutathione reductase, two glutathione-s-transferases, and several kinases. the up-regulation of a putative glutathione reductase together with the consumption of asa suggests that the high asa rils could also have higher asa recycling rates. these results contrast with a previous study reporting that a putative ascorbate peroxidase and a putative glutathione-s-transferase genes were down-regulated in an inbred line selected for its high asa content when compared with the parental line. the different results derived from both studies suggest that asa recycling rate and asa content might be uncoupled events in the tomato rils here employed. the genes down-regulated in the rils group with the highest content of asa could be grouped in clusters. cluster includes the s. lycopersicum 1-aminocyclopropane-1-carboxylate oxidase gene, whose product is an enzyme involved in ethylene biosynthesis that uses asa as a cofactor. the silencing of the acc oxidase gene in tomato is responsible for up to 87% reductions of ethylene in ripening fruits. the acc oxidase down-regulation together with the concomitant down-regulation of a bhlh transcription factor ethylene responsive found in the same cluster seems to point to a reduced ethylene production in the rils with higher asa content. this is further supported by the down-regulation of a gene coding for a s. lycopersicum squamosa promoter binding-like protein in cluster and the s. lycopersicum green ripe-like in cluster spbl is the gene affected in the tomato colorless nonripening mutant that results in colorless fruits with a substantial loss of cell-to-cell adhesion. grl encodes for the closest homolog in tomato of the green ripe gene responsible for the non-ripening phenotype found in the dominant green-ripe tomato mutant. both, spbl and grl are thought to be involved in ethylene responses during tomato fruit ripening, being major regulatory elements in the network controlling the process. in order to discard possible differences in the ripening stage between the two groups of contrasting asa genes that could account for the differential expression of ethylene-related genes we searched for their expression pattern during ripening in the tomato expression database or in the literature. for instance, according to ted, the expression of s. lycopersicum 1-aminocyclopropane-1-carboxylate oxidase gene does not vary significantly between breaker and red stage. on the other hand, it has been reported that the expression of er is induced in red stage and does not vary from mature green to turning, so if the differential expression were due to differences in the ripening stage, they should have occurred in all rils from one group, something highly unlikely. these data allowed us to discard differences in ripening stages as the source of differential expression in ethylene-related genes. clusters and include different elements involved in the responses to gibberellins, such as the s. lycopersicum gibberellin 20-oxidase- gene and the putative gid f-box gene. the gibberellin 20-oxidase- is one of the three ga 20-oxidases involved in the 13-hydroxilation gibberellin biosynthesis pathway. in this pathway, the intermediate ga is converted to ga to be subsequently hydroxilated to the bioactive ga form by ga 3-ox. ga 20-oxidases are main regulatory control points of the gibberellin biosynthesis pathway. the rice gid f-box protein is part of the scf -type e ubiquitin ligase scfgid2/sly that positively regulates gibberellin signaling through the degradation of specific della repressors. the differential regulation of genes implicated in gibberellin biosynthesis, with positive correlation of ga- ox in cluster and negative correlation of ga- ox in cluster is remarkable, opening the possibility that ga influences, points to a non-previously described interaction between gibberellin and asa content in tomato. cluster contains a gene encoding for a putative udp-sugar transporter. how asa transport occurs between compartments of the cell and the apoplast remains largely unknown, as well as the transport of the several intermediates for its synthesis. in many cases, ndp-sugars involved in asa biosynthesis, such as udp-glucose, udp-glucuronic acid and gdp-mannose, are available in the cytosol, and likely require specific transporters to pass through the endomembrane system for further processing. the dow-nregulation of an udp-sugar transporter in the rils containing more asa might be indicative of the importance of ndp-sugar transporters in the final content of asa in a plant tissue. cluster groups two cell wall related genes, a putative cellulose synthase and a xyloglucan endotransglycosylase precursor slxet induced by brassinoesteroids. other cell wall modifying enzyme, the s. lycopersicum endoxyloglucan transferase, was identified in cluster this result might reveal a link between cell wall modification, degradation, and asa content. normally, slxet appears strongly down-regulated at the beginning of turning stage of fruit ripening and appears undetectable at the red stage, in contrast to slxet whose expression drops at the turning and pink stages, to raise again at the red stage. finally, a putative cytochrome p whose expression pattern resembles that of slxet was also identified in cluster cytochromes p many of which are involved in the biosynthesis and metabolism of brassinosteroids, are known to control the redox status of the cell. the fact that several elements controlling the plant redox status were identified in our analysis lead us to hypothesize that this process might be determinant for the asa accumulation in tomato fruit. genes correlated with asa content in tomato fruit are regulated by oxidative stress databases for co-expression analysis have been established for the model plant arabidopsis thaliana, which can be used to get insights into the mechanisms underlying the differential transcript abundance in our contrasting asa lines. as result, various repositories of transcriptome data are now available including nascarrays, geo, smd, arrayexpress, and atgenexpress,, which collectively provide microarray data points. using this repository, we have searched for the most homologous pvc and nvc genes in arabidopsis, and we have analyzed using a correlation analysis of the clustering tools in genevestigator v whether different experimental conditions were reported to cause the transcriptional induction or repression of these sets of genes. the results of our co-expression datasets were analyzed independently for pvc and nvc genes using this huge data set repository. we were expecting that if the pvc and nvc set of genes have a collective biological significance they must be regulated by similar conditions in an opposite way. we identified that % of the atpvc genes were induced by flagellin. importantly, we found that % of atnvc genes were repressed by flag treatment in an independent correlation analysis. the fact that independent correlation analyses using atpvc and atnvc genes rendered similar treatments in arabidopsis clearly indicates that these are relevant biological associations. early events caused by flag treatments include ion fluxes across the plasma membrane and the formation of reactive oxygen species. this relationship of pvc and nvc genes with a ros-generating agent adds further support to the association of asa content and the maintenance of the redox state, as deduced from the clustering analysis.  the differentially expressed genes that correlate with asa content in tomato fruits identified in the present study suggest a scenario where several genes involved in hormone responses are interlinked to modulate the content of asa. cross-talk among different hormones has been reported to regulate several processes in plants: gibberellins and auxin in tomato fruit set; gibberellins and aba during seed germination; gibberellins and ethylene, in root elongation; aba and ethylene, in the modulation the overall carbon status during early seedling growth and development; brassinosteroids and auxin in the control of lateral root growth, etc. ethylene has been implicated indirectly in the regulation of reduced asa content in pericarp prior to tomato ripening as the ethylene-insensitive nr mutant accumulates almost 2-fold asa compared to wild type during mature green and breaker stages. aba has also been connected with the increase of asa content through promoting its recycling. the co-regulation analyses in arabidopsis using the pvc and nvc orthologs identified that these genes are similarly regulated by flag whose primary effect is the generation of ros in response to pathogen attack. these results suggest that asa content in tomato fruit might be critically regulated by hormone interactions, and it is directly related to the oxidative status of the fruit, as previously suggested. however an important aspect that needs to be considered is the likely possibility that part of the asa in the tomato fruit as has been recently reported. therefore part of the fruit asa, in addition of in situ biosynthesis, is likely to be provided by source organs complicating even more the identification of asa determinants. current approaches have been unfruitful in explaining the natural variability of asa content in tomato fruit. the combination of different omics approaches, surveying several metabolites, genes, and gene products seems to be the most realistic way of finding out the source of complex traits like asa content, whose variation among different tomato species is intricately related with hormones. the exhaustive analyses of the omics data provides a more rational picture of the actual physiological processes determining the asa fruit content, and would allow to target sets of co-expressed genes to be further studied by functional analyses. 
l-ascorbic acid is essential for all living plants where it functions as the main hydrosoluble antioxidant. to identify genes responsible for asa content in tomato fruit, transcriptomic studies followed by clustering analysis were applied to two groups of fruits with contrasting asa content. we found large variability in asa content within the ril population with individual rils with up to 4-fold difference in asa content. on the contrary, the data presented here supports that asa content in tomato fruit co-regulates with genes involved in hormone signaling and they are dependent on the oxidative status of the fruit. results here reported indicate that the content of asa in red tomato fruit from our selected rils are not correlated with the expression of genes involved in its biosynthesis. cluster analysis using sota allowed the identification of subsets of co-regulated genes mainly involved in hormones signaling, such as ethylene, aba, gibberellin and auxin, rather than any of the known asa biosynthetic genes. transcriptomic analysis identified genes whose expression correlated either positively or negatively with the asa content of the fruits. data mining of the corresponding pvc and nvc orthologs in arabidopis databases identified flagellin and other ros-producing processes as cues resulting in differential regulation of a high percentage of the genes from both groups of co-regulated genes; more specifically, % of the orthologous pvc genes, and % of the orthologous nvc genes were induced and repressed, respectively, under flagellin treatment in arabidopsis thaliana. asa is also an essential component of the human diet, being tomato fruit one of the main sources of this vitamin. it has diverse roles in the regulation of plant cell growth and expansion, photosynthesis, and hormone-regulated processes.
 l-ascorbic acid is essential for all living plants where it functions as the main hydrosoluble antioxidant. it has diverse roles in the regulation of plant cell growth and expansion, photosynthesis, and hormone-regulated processes. asa is also an essential component of the human diet, being tomato fruit one of the main sources of this vitamin. to identify genes responsible for asa content in tomato fruit, transcriptomic studies followed by clustering analysis were applied to two groups of fruits with contrasting asa content. these fruits were identified after asa profiling of an f recombinant inbred line population generated from a cross between the domesticated species solanum lycopersicum and the wild relative solanum pimpinellifollium.  we found large variability in asa content within the ril population with individual rils with up to 4-fold difference in asa content. transcriptomic analysis identified genes whose expression correlated either positively or negatively with the asa content of the fruits. cluster analysis using sota allowed the identification of subsets of co-regulated genes mainly involved in hormones signaling, such as ethylene, aba, gibberellin and auxin, rather than any of the known asa biosynthetic genes. data mining of the corresponding pvc and nvc orthologs in arabidopis databases identified flagellin and other ros-producing processes as cues resulting in differential regulation of a high percentage of the genes from both groups of co-regulated genes; more specifically, % of the orthologous pvc genes, and % of the orthologous nvc genes were induced and repressed, respectively, under flagellin treatment in arabidopsis thaliana.  results here reported indicate that the content of asa in red tomato fruit from our selected rils are not correlated with the expression of genes involved in its biosynthesis. on the contrary, the data presented here supports that asa content in tomato fruit co-regulates with genes involved in hormone signaling and they are dependent on the oxidative status of the fruit. 