BACKGROUND
peach fruit development is tightly connected to embryogenesis.
fruit growth displays a double sigmoid pattern in which four stages named s <dig>  s <dig>  s <dig> and s <dig> can be distinguished  <cit> .
the early part of s <dig> is characterized by cell division and enlargement lasting about two weeks, followed by cell enlargement.
the slowdown in growth that occurs at s1/s <dig> transition is followed by endocarp lignification , which lasts for 12- <dig> days from the middle of s <dig> to its end.
s <dig> starts with a resumption of growth mainly due to cell enlargement, thus generating the second exponential phase.
maturation is completed by the end of s <dig> and followed by ripening .
the four fruit developmental phases are determined using a mathematical model based on first derivative of the growth curve  <cit> .
identification of the growth phases is important both for developmental studies and for precision farming.
however, the only easily detectable event is the end of pit hardening marking the s2/s <dig> transition, because the phase length is affected by both genotype  and environmental cues.
a continuous growth model reassessment is therefore required.
accordingly, the identification of developmental phase organ-specific molecular markers would be of great importance for scientific and practical purposes.
seed development, necessary for fruit set  <cit> , is characterized by a fast endosperm growth that starts immediately after fertilization concurrently with the nucellus re-absorption, and lasts until the beginning of endocarp lignification, when the seed reaches its final size.
at the end of pit hardening, seed volume is mainly made up of endosperm and the embryo is at the heart stage.
thereafter, embryo growth resumes and cotyledon development is paralleled by endosperm re-absorption.
seed maturation is characterized by lipids accumulations  <cit> , synthesis of specific late embryogenesis abundant  proteins and dehydration.
attempts to stimulate parthenocarpic fruit development by hormone applications resulted as being ineffective.
moreover, seed abnormalities at the early stages of development  lead to abortion and fruitlet abscission  <cit> .
later, , the relationships between fruit development and embryogenesis become less strict.
this is the case for early ripening varieties characterized by the uncoupling of fruit development and embryogenesis.
in fact, at harvest, seed development is still in progress and a long way from maturity.
seed presence is always necessary to achieve normal fruit development even if embryo development is incomplete  <cit> .
apart from the above observations, molecular-genetic information on the relationship between fruit and seed development is scarce.
cross-talk between the two organs may involve different components of the signaling network, such as hormones, transcription factors  and other signaling molecules, playing either direct or indirect roles.
concerning hormones, parthenocarpic fruit development in some species is induced by applications of auxin or cytokinins , or gibberellins , or hormone blends  <cit> .
molecular approaches have confirmed the role played by hormones, especially auxins  <cit> .
investigations in arabidopsis identified a mutant, named fwf , with a normal silique development even in the absence of seeds  <cit> .
double mutant analysis  pointed out that fwf negatively affected ga biosynthesis and ga and auxin signal transduction.
the fwf protein may interact with tfs such as fruitful  and aberrant testa shape , members of the mads-box family, and scarecrow -scr- type, which are all involved in cell division  <cit> .
additional tfs have been identified, some of which are related to hormone action, actively transcribed along peach fruit development and ripening .
orthologues of these tfs are also expressed in true  and false  fruits, supporting the hypothesis that different fruit types share common regulatory elements  <cit> .
high throughput analysis conducted in arabidopsis showed that some tfs are shared by seed and fruit  <cit> .
taking this information into account, peach seed and fruit transcriptomes were explored throughout development by means of a massive gene approach based on the use of the μpeach  <dig>  array platform and quantitative real time rt-pcr .
the research identified genes marking organ/tissue developmental phases, as well as candidate signals  that may trigger the cross-talk between fruit and seed.
RESULTS
seed and fruit growth pattern
fruit growth analysis was performed on cv fantasia and assumed as a reference .
in this genotype fruit development and ripening are completed in 135- <dig> days after full bloom .
growth dynamics display the typical double sigmoidal pattern in which four developmental stages have been identified according to the first derivative.
s <dig>  s <dig>  s <dig> and s <dig> lasted for  <dig>   <dig>   <dig> and  <dig> days, respectively.
pit hardening  started  <dig> dafb and was completed by the s2/s <dig> transition.
the seed derives from the fertilized ovule and the initial increase in length  is due to the rapid nuclear division of the endosperm responsible for embryo sac expansion.
endosperm cellularization starts  <dig> dafb and is completed by the beginning of ph.
the embryo develops very slowly in the early stages , reaching a length of about 40- <dig> μm.
later, at the s2/s <dig> transition, it resumes development reaching its final size by the middle of s <dig>  the morphological completion of development is followed by maturation and desiccation.
identification of marker genes
rnas extracted before  and after  pit hardening have been used for microarray transcriptome analyses in order to identify genes possibly involved in seed-pericarp cross-talk or useful as organ and developmental phase molecular markers.
data obtained from the microarray analyses were handled either as single comparisons, i.e.
late seed vs. early seed , late mesocarp vs. early mesocarp , within each hybridization or by combining the whole set of data, thus also including es/em and ls/lm .
the microarray expression data , validated by means of qrt-pcr on  <dig> randomly selected genes, showed a pearson correlation coefficient ranging, in the four comparisons, from  <dig>  to  <dig>  .
with the single comparison analyses, among the  <dig> differentially expressed genes within the two organs at early and late development ,  <dig> and  <dig> were differentially expressed only in seed  and mesocarp , respectively.
of the seed differentially expressed genes,  <dig> and  <dig> were more transcribed at early  and late development , respectively.
four genes, shared by seed and mesocarp, were more actively transcribed at late development , while an additional four showed the opposite trend of expression, being induced in ls and repressed in lm .
in addition to the  <dig> genes more abundantly transcribed in es ,  <dig> were also expressed in em , while  <dig> were abundant in es and em .
among the mesocarp differentially expressed genes,  <dig> and  <dig> were more transcribed in em  and lm , respectively.
taking the comparison between seed and mesocarp  into account,  <dig> genes were differentially expressed in the two organs .
among these,  <dig> and  <dig> were differentially expressed only at early  and late  development, respectively.
considering the differentially-expressed genes at early development,  <dig> mrnas were more abundant in seed  and  <dig> in mesocarp , while among the late development ones,  <dig> were more abundant in seed  and  <dig> in mesocarp .
of the  <dig> remaining genes,  <dig> and  <dig> transcripts were always more abundant in seed  and mesocarp , respectively, and  <dig> displayed an opposite pattern, being more  or less  abundant in es  and ml .
annotations of genes included in figure  <dig> are reported with microarray expression data in additional file  <dig>
based on the above microarray analysis, putative markers were searched to find those that meet the following criteria: a) moderately to highly expressed in only one organ , b) highly expressed/not expressed at specific developmental stage/s .
according to these criteria,  <dig> potential marker genes, chosen among those differentially expressed in the microarray, were selected and tested by means of qrt-pcr in leaf, flower , seed and mesocarp at five developmental stages in cv fantasia .
these detailed expression profiles allowed the identification of eight genes best fulfilling the ideal marker criteria.
for seed development, ctg <dig>  coding for a lipid transfer protein , ctg <dig>  coding for a pathogenesis related  protein, ctg <dig>  coding for a prunin, and ctg <dig>  coding for a late embryogenesis abundant  protein, have been chosen as s <dig>  s <dig>  s <dig> and s <dig> markers, respectively.
concerning mesocarp development, ctg <dig>  coding for a rd22-like protein, ctg <dig>  coding for serine carboxypeptidase, ctg <dig>  coding for a senescent associated protein, and ctg <dig>  coding for an aux/iaa protein, have been selected as s <dig>  s <dig>  s <dig> and s <dig> markers, respectively .
the function as stage markers has been confirmed on the same genotype for an additional growing season .
a further validation of the selected genes was performed in two additional genotypes  differing for the dynamics of seed and fruit development.
in springcrest, fruit ripening occurred after  <dig> dafb , when seed development was still in progress .
at the end of the growing season , slr showed a fully developed seed , while the mesocarp development was blocked at stage s <dig>
as regards seed markers, ctg <dig>  coding for a ltp, clearly marked the s <dig> stage for both fantasia and slr, while in springcrest its expression decreased only at s <dig> stage .
a pr protein encoding gene, ctg <dig>  has been selected for the s <dig> stage.
the highest expression level was found in the seed of cv fantasia, peaking at early s <dig> and decreasing thereafter, as in springcrest.
in slr, its expression was broader, being relevant also at s <dig> and s2ii .
a prunin, the main seed storage protein in prunus spp., encoded by ctg <dig>  is a good marker for s <dig> seed development only in fantasia.
in fact, different amounts and kinetics of its transcript accumulation were observed in the other two genotypes.
in fantasia, accumulation started between s2i and s2ii and increased up to a maximum at s <dig>  decreasing thereafter, whereas in slr and springcrest transcript accumulation was delayed, becoming detectable at s <dig> in the former and s <dig> in the latter .
the expression of the gene encoding a lea protein  became detectable at s2ii in fantasia and peaked at s <dig>  a similar pattern was observed in slr, although the transcript only started to be detectable at s <dig>  in springcrest, it was detectable only at s <dig>  at levels lower than in the other two genotypes .
the level of expression of the four genes in mesocarp was very low throughout development and comparable in the three cvs .
as regards mesocarp, ctg <dig>  coding for an rd22-like protein, had maximum expression at s <dig> and early s <dig> .
in fantasia and slr the expression decreased already at s2ii , while in springcrest its expression was still high  at s2ii.
a serine carboxypeptidase  was chosen as a marker for the s <dig> developmental stage.
in fantasia, the transcript was undetectable at s <dig>  at basal level at s2i, peaked sharply at s2ii, and then declined at s <dig> and s <dig>  also in the other two varieties the transcript was undetectable at s <dig>  but its expression, already high at s2i, slightly increased at s2ii and remained at high levels at s <dig>  decreasing at s <dig> .
the expression of ctg <dig>  encoding a senescence related protein, had a maximum in fantasia at s <dig> , while expression levels were much lower in the previous and following stages .
although its expression was relatively high  at s2i, it may be considered a good s <dig> marker.
in springcrest, the expression was generally low at all stages, with a maximum at s2ii.
in the slr genotype, the accumulation of ctg <dig> transcripts steadily increased during the early phases up to a maximum at s2ii.
although slightly decreasing thereafter, the ctg <dig> mrna was also abundant at s <dig> and s <dig>  .
s <dig> stage is clearly identified by the expression of ctg <dig>  coding for an aux/iaa protein.
in fantasia, the expression at s <dig> is about 6% of that measured at s <dig> and almost undetectable in early phases.
in springcrest its expression is also almost undetectable at s <dig>  s2i and s2ii, but at s <dig> it is already half of that measured at s <dig>  in slr, although maximum expression is at s <dig>  the transcripts accumulated at very low levels  .
in agreement with their being mesocarp markers, all the selected genes are almost undetectable in seed  with the exception of ctg <dig> in slr.
hormones and tfs in seed fruit cross-talk
hormone-related genes possibly involved in cross-talk between the two organs were identified among those spotted on the microarray based upon the list of hormonal indexes available for arabidopsis .
the portion of hormone responsive genes in arabidopsis ranges between  <dig>  and  <dig> % of the whole transcriptome , depending on the hormone considered .
for μpeach  <dig>  , the portion of hormone responsive genes parallels that of arabidopsis, ranging from  <dig>  to  <dig> % with values for each hormone class comparable to those calculated for arabidopsis.
an irrelevant bias may therefore be assumed to exist when peach expression data are used for hormonometer analysis  <cit> .
in addition, it could be assumed that the same proportion might be expected if a whole genome array were used.
a heat map was produced by considering the following subsets of genes for each hormone : i) genes involved in signal transduction , ii) hormone-responsive genes , iii) genes with hormone-specific responsiveness , iv) hormone-responsive genes encoding tfs , and v) genes encoding tfs with hormone-specific responsiveness .
the subset i) was identified using the classification of arabidopsis orthologs obtained from tair go terms and ahd classification lists , and was then analyzed by averaging the log ratios, while the other subsets were used for the hormonometer analyses  <cit> .
concerning auxin and intra-organ comparisons , a weak activation of st was observed in ls with respect to es, paralleled by a partial correlation with the overall reference hormone indexes, whereas a partial anti-correlation was observed when auxin-specific hormone indexes, tf- and specific tf-encoding targets were used.
in the mesocarp, a marked up-regulation of st subset was evidenced in lm, and a good correlation was shown in the same sample both considering the overall hormone indexes and all the other gene subsets.
as regards inter-organ comparisons, a decreased transcription of st elements was always observed in the seed, paralleled by an anti-correlation with the overall hormone indexes at both early  and late  development.
however, considering the specific subset, a slight correlation was found in the former comparison, whereas all the results in the latter one were consistent with the overall hormonometer data.
the intra-organ comparison ls/es indicated a down-regulation of cytokinin  st elements at late seed development, paralleled by an anti-correlation with both the overall and specific hormone indexes.
however, a slight correlation was observed in terms of specific tfs, while all tfs appeared not correlated.
concerning the mesocarp, a lower activation of st elements in lm than em was counteracted by a strong correlation with ck indexes.
ck-specific genes appeared not correlated, whereas tfs showed a slight correlation, becoming stronger when only the ck-specific tfs were considered.
as regards inter-organ comparisons, a low activation of the signal transduction in es was counteracted by a strong correlation with overall hormone indexes.
when the analysis was performed with the other subsets, a significant anti-correlation was observed.
finally, during late seed development, despite the higher activation of st elements compared to the mesocarp, a general anti-correlation was shown, with the exception of specific tfs, which appeared not correlated.
considering the gibberellins -related expression data, the ls/es comparison demonstrated a good consistency in signal transduction, and anti-correlation with overall and specific transcriptional indexes, and tfs, except for the ga-specific tfs, that were not correlated.
the mesocarp profile was similar except when all tfs were considered, the latter analysis showing a robust correlation.
in the es/em inter-organ comparison, a depression of the st pathway in the seed was evidenced.
the overall hormonometer analysis showed no correlation with ga hormone indexes, whereas an anti-correlation resulted from the analysis of hormone-specific targets.
when all the tfs underwent the hormonometer analysis, a strong correlation was shown, while specific tfs were not correlated.
the most significant data pointed out by the ls/lm comparison concerned the analysis of ga-specific indexes, showing a slight correlation.
as regards abscisic acid  and intra-organ comparisons, in spite of a down-regulation of its st pathway during late seed development, a correlation was observed in terms of both overall and aba-specific indexes.
tfs were basically anti-correlated and not correlated, when considered either as a whole or just the specific ones, respectively.
in the mesocarp, despite a weak up-regulation of the st elements found in lm, there was no significant correlation in any of the hormonometer analyses.
moving to inter-organ comparison es/em, the down-regulation of signal transduction occurring in es paralleled an anti-correlation found in all the gene sets.
in the ls/lm comparison, similar results were obtained in terms of both signal transduction and hormonometer.
concerning ethylene, no variation was observed between ls and es in terms of expression of genes encoding st elements.
in spite of this, a slight correlation was pointed out by both overall and ethylene-specific gene targets.
moreover, tfs were not correlated, while specific tfs were slightly anti-correlated.
with the lm/em comparison, the hormone signaling pathway was up-regulated in lm, paralleled by a partial correlation of tfs.
on the other hand, both the hormone specific subsets showed an anti-correlation, stronger in the case of tfs.
both inter-organ comparisons  displayed a down-regulation of the st pathway in the seed.
the hormonometer analyses showed no correlation when all targets and all tfs were considered, and anti-correlation concerning the specific targets and tfs, stronger for the former.
both signal transduction and hormonometer results related to jasmonates, salicylic acid, and brassinosteroids are presented and discussed in additional file  <dig>
discussion
this research was mainly focused on the relationship between seed and pericarp throughout development, using a mass gene approach by means of the μpeach <dig>   <cit> .
although this platform was developed mainly from late development mesocarp cdnas, hybridization analyses and differential expression profiles assessed for both early developing mesocarp and seed indicate that μpeach <dig>  is also a reliable tool for these transcriptomic investigations.
concerning marker genes, morphological observations pointed out that the dynamics of seed development in different genotypes is quite synchronous, whereas a wide variation exists in the pericarp, affecting not only the length of the developmental phases but also important traits related to fruit quality, such as the degree of endocarp lignification , flesh texture , sugar/acid ratio, etc.
accordingly, the singling out of marker genes specific for the same developmental stage is not always unequivocal for all three studied genotypes.
moreover, since seed sampling was referred to the fruit developmental stages , expression data should be read taking into account the uncoupling that exists between seed and fruit development in springcrest, an early ripening cultivar.
the ctg <dig>  marking s <dig> in the seed, encodes a lipid transfer protein similar to arabidopsis ltp <dig>  <cit> .
its gene expression profile in peach is consistent with arabidopsis data, the latter showing that ltp <dig>  along with ltp <dig>  ltp <dig> and ltp <dig>  is expressed at high levels during early seed development  <cit> .
the function of this gene as an s <dig> marker was confirmed in all the genotypes.
the delayed decay of transcript accumulation assessed in the seed of cv springcrest has, in fact, to be related to the acceleration of mesocarp development in this genotype .
the ctg <dig>  is similar to a carrot pr which has been related to early embryo development, being expressed in the endosperm and secreted in the apoplast, thus positively regulating embryo fate and patterning  <cit> .
it is interesting to note that in cv springcrest, the down-regulation of ctg <dig> at s <dig> and s <dig> occurs at a slower rate than in fantasia and slr, thus confirming the uncoupling of seed and mesocarp development also at the molecular level.
the different kinetics observed for the expression of s <dig> marker, a gene encoding a prunin storage protein  in slr indicates that in this selection, as well as the blocked development of the mesocarp, some variations in seed storage accumulation may also exist.
the apparent delay in transcript accumulation measured in springcrest is again due, as in the case of ctg <dig>  to the uncoupled development of seed and pericarp.
ctg <dig>  encoding a lea  protein, is a very reliable marker of s <dig>  in both fantasia and slr, indicating that the seed can reach a fully matured stage in both genotypes.
the very low levels of lea gene expression detected at s <dig> in springcrest are consistent with the uncoupling that exists between seed and pericarp maturation in this genotype.
concerning the mesocarp, ctg <dig>  marking s <dig> and s2i, encodes a putative rd22-like protein, whose expression in both arabidopsis and grape is partially under the control of aba and claimed to be involved in stress responses  <cit> .
since the levels of this hormone in peach mesocarp were shown to follow a biphasic pattern with two peaks at s2i and s <dig>  <cit> , the increasing expression of ctg <dig> at early mesocarp development might be related to the level of aba.
however, while the hormone also peaks at s <dig>  the expression levels of this gene did not, thus indicating a dual regulatory mechanism triggering its expression, possibly also under a developmental control as shown in the seed of arabidopsis  <cit> .
the delayed decay of ctg <dig> expression observed in springcrest might be related to the higher growth potential of this early ripening variety documented by the s <dig> phase length, which is significantly reduced compared to cv fantasia .
the s <dig> phase is marked by ctg <dig> , coding for a serine carboxypeptidase .
scps are members of the α/β hydrolase family of proteins, claimed to function also as acyltransferases and lyases in the biosynthesis of secondary metabolites  <cit> .
taking into account that the most important event occurring at s2ii is endocarp lignification, an indirect role in this process might be hypothesized for ctg <dig>  ctg <dig>  was shown to be a good s <dig> marker in fantasia, but not in slr and springcrest.
since this gene encodes a putative senescence-associated protein, a likely failure of the senescence process and/or of its entry phase may be hypothesized in the two genotypes in which mesocarp development is either slowed down or accelerated.
interestingly, when mesocarp development is slowed down, as in slr, the peak of expression of ctg <dig> is anticipated, whereas in the other case , in which mesocarp ripens very rapidly, the peak is almost absent.
it may be speculated that an overly precocious start of senescence would not allow the fruit to shift from maturation to ripening  <cit> , and, vice versa, an acceleration of fruit ripening is achieved if senescence is not initiated.
for the s <dig> phase, a very reliable marker is represented by ctg <dig> , coding for an already partially characterized peach aux/iaa protein  <cit> .
its expression was shown to increase at early s <dig>  most likely under a developmental control, thereafter decreasing when ethylene climacteric is fully installed.
accordingly, ethylene treatments were shown to reduce the specific transcripts.
besides fully agreeing with previous data, the expression profiles shown here may also represent correlative evidence for a putative functional role.
indeed, no rise of expression was measured in the mesocarp of slr, consistent with the block/slowdown of development and ripening.
moreover, in the case of springcrest, a high ethylene-producing variety  <cit> , the rise in expression of ctg <dig> is both anticipated, paralleling ripening kinetics, and less pronounced than in fantasia, in agreement with a negative effect exerted by higher levels of ethylene.
possible mechanisms involved in seed-pericarp cross-talk should take into account the vascular and cellular connections existing between the two organs.
it has been shown that all the maternal tissues of pericarp and seed  are intensively interconnected , while nucellar tissue is excluded from the plasmodesmata network.
this implies that the flux of metabolites, as well as signaling molecules between embryo and fruit, must occur through the apoplast.
taking into account that hormones play a pivotal role in the regulation of seed and fruit development, it has been assumed that they might also be involved in the cross-talk between the two organs.
the heat map data  will therefore be discussed taking into account the consistency of the colors in the following main two-by-two comparisons: st/h, srg/h, tfs/h, and stfs/srg.
more specifically, considering the first one , consistency of colors may indicate a relationship between the hormone-related response and activation of the corresponding signal transduction pathway.
in the second comparison , the same parameter may provide information about the hormone specificity of the transcriptional response, and, at the same time, of the possible cross-talk between hormones.
a double comparison  may allow it to be pointed out if other players besides the tfs are involved in the regulation of the downstream processes, and if a specific response is mediated by hormone-specific tfs.
auxin, cytokinins, and gibberellins are generally considered to be the most relevant hormones for early seed and fruit development, whereas abscisic acid and ethylene play important roles in seed maturation and fruit ripening.
from the point of view of the cross-talk between seed and mesocarp, comparisons should refer to the same developmental stage, i.e.
es/em and ls/lm.
concerning auxin, the data presented here point out that the specificity of the response to the hormone is higher in es and lm, although the relationship between the overall hormonometer  and st data indicates that mesocarp is always more sensitive than the seed to the hormone.
taking into account that the presence of a viable seed is required for fruit set and development in peach  <cit> , and that the overexpression of auxin biosynthetic genes in the ovary stimulates the parthenocarpic fruit development in several species  <cit> , it may be hypothesized that the signal produced by the developing seed might be either the auxin itself exported to the fruit, as demonstrated in tomato  <cit> , or a secondary messenger whose target at the fruit level includes a large subset of auxin-responsive genes.
this is consistent with both the high specificity of the auxin response shown here in the early developing seed and the higher sensitivity to the hormone displayed by the mesocarp paralleled by a strong hormone response.
among the mesocarp auxin responsive genes, several encode elements regulating transport , indicating that auxin movement in this tissue is a relevant process, thus strengthening the hypothesis that auxin produced by the seed may behave as a signal efficiently transported to and within the mesocarp.
an aux/iaa-encoding gene  showed an opposite transcription profile in the two organs, being abundant in es and lm.
it has been demonstrated that its tomato orthologue  acts as a repressor of auxin signaling.
thus, its expression in young organs  seems to confirm that the hormonal response is not at the synthesis site.
finally, the expression of ctg <dig>  a saur-like iaa responsive gene  <cit>  was found to be higher in mesocarp than in seed , thus suggesting a higher auxin level in em than in es  <cit> .
the main process regulated by cks is cell division, occurring at early development in both seed  and mesocarp.
in the former, there is an up-regulation of signal transduction elements, such as ctg <dig> coding for a histidine-containing phosphotransfer protein  <cit>  whose transcription is abundant in very young organs .
the corresponding substantial activation of hormonal targets, including several ck-specific genes, might differ in the two organs.
for example, a cellulose synthase  is activated in em but not in es, cytokinesis being an ls event, whereas cyclin d <dig>  was up-regulated in both organs at the early stage.
however, this transcriptional response did not just involve ck-specific tfs, implying that other regulatory elements may determine the hormone-specific gene activation.
a similar activation of signal transduction elements to that found in the seed is present in the mesocarp at early development.
however, the overall and the ck-specific target activation are not correlated to the hormone action, suggesting that cks may regulate mesocarp cell division at the post-transcriptional level  <cit> , either alone or in cooperation with other phytohormones.
moreover, considering the inter-organ comparison, it is noteworthy that during early development the seed displayed a higher sensitivity to cks than the mesocarp but a lower specificity of response.
the amount of the overall transcriptional response observed in the seed may be due to the involvement of other hormones besides cks  <cit> .
during late development, an inverse situation was observed compared to the early phases.
in fact, the high activation of signal transduction pathways occurring in the seed was uncoupled from the overall transcriptional response, which was even more specific in the mesocarp.
the ck-mediated up-regulation of genes encoding sorbitol dehydrogenases  appears particularly interesting, as this might increase the sink strength of the seed and attract photoassimilates to the entire fruit, which become more competitive in the partitioning process  <cit> .
from a physiological point of view, gas play a stimulatory role in fruit development, as shown by the ability to induce parthenocarpy in several species  <cit>  when applied in post-bloom phase and/or early development.
the initial phases of endosperm and embryo development are usually related to a high level of gas  <cit> , while seed maturation is paralleled by a decay of free gas and increase of their conjugates.
the hormonometer data confirmed these results both in seed and mesocarp, except for tfs in the latter.
in fact, the most relevant transcriptional response occurred during early development at seed level as pointed out by the es-specific expression of ctg <dig>  encoding an orthologue of the arabidopsis ltp <dig> , which is classified as a ga-responsive gene  involved in embryo patterning  <cit> .
in the mesocarp, a low correlation was observed between the tf-related transcriptional response and ga action, implying the activation of complex regulatory mechanisms that may play relevant roles in the cross-talk between seed and mesocarp.
a possible mode of interaction might be the em specific expression of a gene coding for a zinc finger protein , whose arabidopsis orthologue  interacts with della proteins, is repressed by ga, and causes aba accumulation when over-expressed  <cit> .
however, since this transcriptional response lacked specificity, it might be hypothesized that ga action also depends on the interaction with other hormones.
it has recently been demonstrated that auxin induced parthenocarpy via gas in unpollinated tomato ovaries  <cit> .
furthermore, the peculiar expression profile of ctg <dig>  encoding a gast-like protein, orthologue of arabidopsis gasa <dig> , in em is confined to s <dig> and s <dig> stages, when cell enlargement is slow .
these data are in agreement with the observed inhibition of cell elongation conferred on both arabidopsis seedling and strawberry fruit over-expressing the fragaria orthologue fagast  <cit> .
during late development, in spite of the slight correlation existing in terms of ga-specific response, the other gene sets appeared not to be correlated to the hormone action.
it may be deduced from this that the role of ga in the cross-talk between seed and mesocarp is negligible during late development.
aba is known to play an antagonistic role with respect to auxin, gas and cks, as observed during fruit development in avocado  <cit>  and tomato  <cit> .
according to the hormonometer, this antagonism was largely confirmed in the seed, the transcriptional response being correlated with higher levels of the hormone in ls compared to es, also when the aba-specific subset was considered.
in fact, during late seed development, aba levels are known to increase and ga-related genes such as ctg <dig>  encoding a ltp-like, are down-regulated .
this physiological parameter is paralleled by a consistent transcriptional response in which tfs belonging to wrky , hd , aux/iaa , bzip  and dreb-like ap <dig>  families are involved.
given this interpretation and taking into account that during both early and late development aba st pathways and aba-target responses are more active in the mesocarp, the hormone may play a more relevant role in the development of each organ, rather than in seed-mesocarp cross-talk.
in this context, the aba pool of maternal and zygotic origin may trigger independent transduction pathways.
the well-known role of ethylene in peach ripening  <cit>  was confirmed by the higher level of transcription of its st elements  measured at the mesocarp level during late development.
it is worth noting that ethylene-related transcriptional response in the lm/em comparison resembles that of aba, most likely because of the significant number of transcriptional targets shared by the two hormones .
this was not observed in the inter-organ assessments, in which, differently from aba, a weaker overall transcriptional response was pointed out, with the only exception being ethylene-specific tfs that were more represented in mesocarp.
as regards cross-talk, the role of ethylene might be limited to the very early phase of fruit development, as demonstrated in tomato  <cit> .
this might be a consequence of the fact that ethylene acts mainly within the cell where it is synthesized.
also in this case, the hormone of maternal and zygotic origin may activate independent pathways controlling different processes.
CONCLUSIONS
in this research, genes were identified marking different developmental phases of seed and mesocarp.
the reliability of these molecular markers was tested in two subsequent years and a further functional validation was carried out in three different genotypes.
in the latter case, data indicate that, while seed markers represent reliable tools in all the tested varieties, in the case of the mesocarp the different developmental and ripening traits of the various genotypes somewhat affect the expression of marker genes, consistently with their putative functions and cv characteristics.
the most critical phases, from the point of view of mesocarp marker retrieval, were s2ii and s <dig>  this might be related to the high divergence in pericarp development among the different genotypes, as pointed out above.
however, this limitation may be partially overcome by using mesocarp markers as a whole, therefore increasing their discriminating power.
as regards the cross-talk between seed and pericarp, possible candidate signals were identified among hormones.
in the early phases, when the cross-talk is more vital for fruit set, the candidates are auxin, cks, and gas, acting either directly  or indirectly as signals, whereas aba and ethylene appear to be involved later on.
further investigations relying upon the availability of whole genome platforms will allow enrichment of the marker genes repertoire and elucidation of the cross-talk mechanisms between the two organs, taking into account, besides hormones, other players such as hormone peptides and micrornas.

 peach fruit development is tightly connected to embryogenesis. fruit growth displays a double sigmoid pattern in which four stages named s s s and s can be distinguished. the early part of s is characterized by cell division and enlargement lasting about two weeks, followed by cell enlargement. the slowdown in growth that occurs at s1/s transition is followed by endocarp lignification, which lasts for 12- days from the middle of s to its end. s starts with a resumption of growth mainly due to cell enlargement, thus generating the second exponential phase. maturation is completed by the end of s and followed by ripening. the four fruit developmental phases are determined using a mathematical model based on first derivative of the growth curve. identification of the growth phases is important both for developmental studies and for precision farming. however, the only easily detectable event is the end of pit hardening marking the s2/s transition, because the phase length is affected by both genotype and environmental cues. a continuous growth model reassessment is therefore required. accordingly, the identification of developmental phase organ-specific molecular markers would be of great importance for scientific and practical purposes. seed development, necessary for fruit set, is characterized by a fast endosperm growth that starts immediately after fertilization concurrently with the nucellus re-absorption, and lasts until the beginning of endocarp lignification, when the seed reaches its final size. at the end of pit hardening, seed volume is mainly made up of endosperm and the embryo is at the heart stage. thereafter, embryo growth resumes and cotyledon development is paralleled by endosperm re-absorption. seed maturation is characterized by lipids accumulations, synthesis of specific late embryogenesis abundant proteins and dehydration. attempts to stimulate parthenocarpic fruit development by hormone applications resulted as being ineffective. moreover, seed abnormalities at the early stages of development lead to abortion and fruitlet abscission. later,, the relationships between fruit development and embryogenesis become less strict. this is the case for early ripening varieties characterized by the uncoupling of fruit development and embryogenesis. in fact, at harvest, seed development is still in progress and a long way from maturity. seed presence is always necessary to achieve normal fruit development even if embryo development is incomplete. apart from the above observations, molecular-genetic information on the relationship between fruit and seed development is scarce. cross-talk between the two organs may involve different components of the signaling network, such as hormones, transcription factors and other signaling molecules, playing either direct or indirect roles. concerning hormones, parthenocarpic fruit development in some species is induced by applications of auxin or cytokinins, or gibberellins, or hormone blends. molecular approaches have confirmed the role played by hormones, especially auxins. investigations in arabidopsis identified a mutant, named fwf, with a normal silique development even in the absence of seeds. double mutant analysis pointed out that fwf negatively affected ga biosynthesis and ga and auxin signal transduction. the fwf protein may interact with tfs such as fruitful and aberrant testa shape, members of the mads-box family, and scarecrow -scr- type, which are all involved in cell division. additional tfs have been identified, some of which are related to hormone action, actively transcribed along peach fruit development and ripening. orthologues of these tfs are also expressed in true and false fruits, supporting the hypothesis that different fruit types share common regulatory elements. high throughput analysis conducted in arabidopsis showed that some tfs are shared by seed and fruit. taking this information into account, peach seed and fruit transcriptomes were explored throughout development by means of a massive gene approach based on the use of the μpeach array platform and quantitative real time rt-pcr. the research identified genes marking organ/tissue developmental phases, as well as candidate signals that may trigger the cross-talk between fruit and seed.  seed and fruit growth pattern fruit growth analysis was performed on cv fantasia and assumed as a reference. in this genotype fruit development and ripening are completed in 135- days after full bloom. growth dynamics display the typical double sigmoidal pattern in which four developmental stages have been identified according to the first derivative. s s s and s lasted for and days, respectively. pit hardening started dafb and was completed by the s2/s transition. the seed derives from the fertilized ovule and the initial increase in length is due to the rapid nuclear division of the endosperm responsible for embryo sac expansion. endosperm cellularization starts dafb and is completed by the beginning of ph. the embryo develops very slowly in the early stages, reaching a length of about 40- μm. later, at the s2/s transition, it resumes development reaching its final size by the middle of s the morphological completion of development is followed by maturation and desiccation. identification of marker genes rnas extracted before and after pit hardening have been used for microarray transcriptome analyses in order to identify genes possibly involved in seed-pericarp cross-talk or useful as organ and developmental phase molecular markers. data obtained from the microarray analyses were handled either as single comparisons, i.e. late seed vs. early seed, late mesocarp vs. early mesocarp, within each hybridization or by combining the whole set of data, thus also including es/em and ls/lm. the microarray expression data, validated by means of qrt-pcr on randomly selected genes, showed a pearson correlation coefficient ranging, in the four comparisons, from to. with the single comparison analyses, among the differentially expressed genes within the two organs at early and late development, and were differentially expressed only in seed and mesocarp, respectively. of the seed differentially expressed genes, and were more transcribed at early and late development, respectively. four genes, shared by seed and mesocarp, were more actively transcribed at late development, while an additional four showed the opposite trend of expression, being induced in ls and repressed in lm. in addition to the genes more abundantly transcribed in es, were also expressed in em, while were abundant in es and em. among the mesocarp differentially expressed genes, and were more transcribed in em and lm, respectively. taking the comparison between seed and mesocarp into account, genes were differentially expressed in the two organs. among these, and were differentially expressed only at early and late development, respectively. considering the differentially-expressed genes at early development, mrnas were more abundant in seed and in mesocarp, while among the late development ones, were more abundant in seed and in mesocarp. of the remaining genes, and transcripts were always more abundant in seed and mesocarp, respectively, and displayed an opposite pattern, being more or less abundant in es and ml. annotations of genes included in figure are reported with microarray expression data in additional file based on the above microarray analysis, putative markers were searched to find those that meet the following criteria: a) moderately to highly expressed in only one organ, b) highly expressed/not expressed at specific developmental stage/s. according to these criteria, potential marker genes, chosen among those differentially expressed in the microarray, were selected and tested by means of qrt-pcr in leaf, flower, seed and mesocarp at five developmental stages in cv fantasia. these detailed expression profiles allowed the identification of eight genes best fulfilling the ideal marker criteria. for seed development, ctg coding for a lipid transfer protein, ctg coding for a pathogenesis related protein, ctg coding for a prunin, and ctg coding for a late embryogenesis abundant protein, have been chosen as s s s and s markers, respectively. concerning mesocarp development, ctg coding for a rd22-like protein, ctg coding for serine carboxypeptidase, ctg coding for a senescent associated protein, and ctg coding for an aux/iaa protein, have been selected as s s s and s markers, respectively. the function as stage markers has been confirmed on the same genotype for an additional growing season. a further validation of the selected genes was performed in two additional genotypes differing for the dynamics of seed and fruit development. in springcrest, fruit ripening occurred after dafb, when seed development was still in progress. at the end of the growing season, slr showed a fully developed seed, while the mesocarp development was blocked at stage s as regards seed markers, ctg coding for a ltp, clearly marked the s stage for both fantasia and slr, while in springcrest its expression decreased only at s stage. a pr protein encoding gene, ctg has been selected for the s stage. the highest expression level was found in the seed of cv fantasia, peaking at early s and decreasing thereafter, as in springcrest. in slr, its expression was broader, being relevant also at s and s2ii. a prunin, the main seed storage protein in prunus spp., encoded by ctg is a good marker for s seed development only in fantasia. in fact, different amounts and kinetics of its transcript accumulation were observed in the other two genotypes. in fantasia, accumulation started between s2i and s2ii and increased up to a maximum at s decreasing thereafter, whereas in slr and springcrest transcript accumulation was delayed, becoming detectable at s in the former and s in the latter. the expression of the gene encoding a lea protein became detectable at s2ii in fantasia and peaked at s a similar pattern was observed in slr, although the transcript only started to be detectable at s in springcrest, it was detectable only at s at levels lower than in the other two genotypes. the level of expression of the four genes in mesocarp was very low throughout development and comparable in the three cvs. as regards mesocarp, ctg coding for an rd22-like protein, had maximum expression at s and early s. in fantasia and slr the expression decreased already at s2ii, while in springcrest its expression was still high at s2ii. a serine carboxypeptidase was chosen as a marker for the s developmental stage. in fantasia, the transcript was undetectable at s at basal level at s2i, peaked sharply at s2ii, and then declined at s and s also in the other two varieties the transcript was undetectable at s but its expression, already high at s2i, slightly increased at s2ii and remained at high levels at s decreasing at s. the expression of ctg encoding a senescence related protein, had a maximum in fantasia at s, while expression levels were much lower in the previous and following stages. although its expression was relatively high at s2i, it may be considered a good s marker. in springcrest, the expression was generally low at all stages, with a maximum at s2ii. in the slr genotype, the accumulation of ctg transcripts steadily increased during the early phases up to a maximum at s2ii. although slightly decreasing thereafter, the ctg mrna was also abundant at s and s. s stage is clearly identified by the expression of ctg coding for an aux/iaa protein. in fantasia, the expression at s is about 6% of that measured at s and almost undetectable in early phases. in springcrest its expression is also almost undetectable at s s2i and s2ii, but at s it is already half of that measured at s in slr, although maximum expression is at s the transcripts accumulated at very low levels. in agreement with their being mesocarp markers, all the selected genes are almost undetectable in seed with the exception of ctg in slr. hormones and tfs in seed fruit cross-talk hormone-related genes possibly involved in cross-talk between the two organs were identified among those spotted on the microarray based upon the list of hormonal indexes available for arabidopsis. the portion of hormone responsive genes in arabidopsis ranges between and % of the whole transcriptome, depending on the hormone considered. for μpeach, the portion of hormone responsive genes parallels that of arabidopsis, ranging from to % with values for each hormone class comparable to those calculated for arabidopsis. an irrelevant bias may therefore be assumed to exist when peach expression data are used for hormonometer analysis. in addition, it could be assumed that the same proportion might be expected if a whole genome array were used. a heat map was produced by considering the following subsets of genes for each hormone: i) genes involved in signal transduction, ii) hormone-responsive genes, iii) genes with hormone-specific responsiveness, iv) hormone-responsive genes encoding tfs, and v) genes encoding tfs with hormone-specific responsiveness. the subset i) was identified using the classification of arabidopsis orthologs obtained from tair go terms and ahd classification lists, and was then analyzed by averaging the log ratios, while the other subsets were used for the hormonometer analyses. concerning auxin and intra-organ comparisons, a weak activation of st was observed in ls with respect to es, paralleled by a partial correlation with the overall reference hormone indexes, whereas a partial anti-correlation was observed when auxin-specific hormone indexes, tf- and specific tf-encoding targets were used. in the mesocarp, a marked up-regulation of st subset was evidenced in lm, and a good correlation was shown in the same sample both considering the overall hormone indexes and all the other gene subsets. as regards inter-organ comparisons, a decreased transcription of st elements was always observed in the seed, paralleled by an anti-correlation with the overall hormone indexes at both early and late development. however, considering the specific subset, a slight correlation was found in the former comparison, whereas all the results in the latter one were consistent with the overall hormonometer data. the intra-organ comparison ls/es indicated a down-regulation of cytokinin st elements at late seed development, paralleled by an anti-correlation with both the overall and specific hormone indexes. however, a slight correlation was observed in terms of specific tfs, while all tfs appeared not correlated. concerning the mesocarp, a lower activation of st elements in lm than em was counteracted by a strong correlation with ck indexes. ck-specific genes appeared not correlated, whereas tfs showed a slight correlation, becoming stronger when only the ck-specific tfs were considered. as regards inter-organ comparisons, a low activation of the signal transduction in es was counteracted by a strong correlation with overall hormone indexes. when the analysis was performed with the other subsets, a significant anti-correlation was observed. finally, during late seed development, despite the higher activation of st elements compared to the mesocarp, a general anti-correlation was shown, with the exception of specific tfs, which appeared not correlated. considering the gibberellins -related expression data, the ls/es comparison demonstrated a good consistency in signal transduction, and anti-correlation with overall and specific transcriptional indexes, and tfs, except for the ga-specific tfs, that were not correlated. the mesocarp profile was similar except when all tfs were considered, the latter analysis showing a robust correlation. in the es/em inter-organ comparison, a depression of the st pathway in the seed was evidenced. the overall hormonometer analysis showed no correlation with ga hormone indexes, whereas an anti-correlation resulted from the analysis of hormone-specific targets. when all the tfs underwent the hormonometer analysis, a strong correlation was shown, while specific tfs were not correlated. the most significant data pointed out by the ls/lm comparison concerned the analysis of ga-specific indexes, showing a slight correlation. as regards abscisic acid and intra-organ comparisons, in spite of a down-regulation of its st pathway during late seed development, a correlation was observed in terms of both overall and aba-specific indexes. tfs were basically anti-correlated and not correlated, when considered either as a whole or just the specific ones, respectively. in the mesocarp, despite a weak up-regulation of the st elements found in lm, there was no significant correlation in any of the hormonometer analyses. moving to inter-organ comparison es/em, the down-regulation of signal transduction occurring in es paralleled an anti-correlation found in all the gene sets. in the ls/lm comparison, similar results were obtained in terms of both signal transduction and hormonometer. concerning ethylene, no variation was observed between ls and es in terms of expression of genes encoding st elements. in spite of this, a slight correlation was pointed out by both overall and ethylene-specific gene targets. moreover, tfs were not correlated, while specific tfs were slightly anti-correlated. with the lm/em comparison, the hormone signaling pathway was up-regulated in lm, paralleled by a partial correlation of tfs. on the other hand, both the hormone specific subsets showed an anti-correlation, stronger in the case of tfs. both inter-organ comparisons displayed a down-regulation of the st pathway in the seed. the hormonometer analyses showed no correlation when all targets and all tfs were considered, and anti-correlation concerning the specific targets and tfs, stronger for the former. both signal transduction and hormonometer results related to jasmonates, salicylic acid, and brassinosteroids are presented and discussed in additional file discussion this research was mainly focused on the relationship between seed and pericarp throughout development, using a mass gene approach by means of the μpeach. although this platform was developed mainly from late development mesocarp cdnas, hybridization analyses and differential expression profiles assessed for both early developing mesocarp and seed indicate that μpeach is also a reliable tool for these transcriptomic investigations. concerning marker genes, morphological observations pointed out that the dynamics of seed development in different genotypes is quite synchronous, whereas a wide variation exists in the pericarp, affecting not only the length of the developmental phases but also important traits related to fruit quality, such as the degree of endocarp lignification, flesh texture, sugar/acid ratio, etc. accordingly, the singling out of marker genes specific for the same developmental stage is not always unequivocal for all three studied genotypes. moreover, since seed sampling was referred to the fruit developmental stages, expression data should be read taking into account the uncoupling that exists between seed and fruit development in springcrest, an early ripening cultivar. the ctg marking s in the seed, encodes a lipid transfer protein similar to arabidopsis ltp. its gene expression profile in peach is consistent with arabidopsis data, the latter showing that ltp along with ltp ltp and ltp is expressed at high levels during early seed development. the function of this gene as an s marker was confirmed in all the genotypes. the delayed decay of transcript accumulation assessed in the seed of cv springcrest has, in fact, to be related to the acceleration of mesocarp development in this genotype. the ctg is similar to a carrot pr which has been related to early embryo development, being expressed in the endosperm and secreted in the apoplast, thus positively regulating embryo fate and patterning. it is interesting to note that in cv springcrest, the down-regulation of ctg at s and s occurs at a slower rate than in fantasia and slr, thus confirming the uncoupling of seed and mesocarp development also at the molecular level. the different kinetics observed for the expression of s marker, a gene encoding a prunin storage protein in slr indicates that in this selection, as well as the blocked development of the mesocarp, some variations in seed storage accumulation may also exist. the apparent delay in transcript accumulation measured in springcrest is again due, as in the case of ctg to the uncoupled development of seed and pericarp. ctg encoding a lea protein, is a very reliable marker of s in both fantasia and slr, indicating that the seed can reach a fully matured stage in both genotypes. the very low levels of lea gene expression detected at s in springcrest are consistent with the uncoupling that exists between seed and pericarp maturation in this genotype. concerning the mesocarp, ctg marking s and s2i, encodes a putative rd22-like protein, whose expression in both arabidopsis and grape is partially under the control of aba and claimed to be involved in stress responses. since the levels of this hormone in peach mesocarp were shown to follow a biphasic pattern with two peaks at s2i and s, the increasing expression of ctg at early mesocarp development might be related to the level of aba. however, while the hormone also peaks at s the expression levels of this gene did not, thus indicating a dual regulatory mechanism triggering its expression, possibly also under a developmental control as shown in the seed of arabidopsis. the delayed decay of ctg expression observed in springcrest might be related to the higher growth potential of this early ripening variety documented by the s phase length, which is significantly reduced compared to cv fantasia. the s phase is marked by ctg, coding for a serine carboxypeptidase. scps are members of the α/β hydrolase family of proteins, claimed to function also as acyltransferases and lyases in the biosynthesis of secondary metabolites. taking into account that the most important event occurring at s2ii is endocarp lignification, an indirect role in this process might be hypothesized for ctg ctg was shown to be a good s marker in fantasia, but not in slr and springcrest. since this gene encodes a putative senescence-associated protein, a likely failure of the senescence process and/or of its entry phase may be hypothesized in the two genotypes in which mesocarp development is either slowed down or accelerated. interestingly, when mesocarp development is slowed down, as in slr, the peak of expression of ctg is anticipated, whereas in the other case, in which mesocarp ripens very rapidly, the peak is almost absent. it may be speculated that an overly precocious start of senescence would not allow the fruit to shift from maturation to ripening, and, vice versa, an acceleration of fruit ripening is achieved if senescence is not initiated. for the s phase, a very reliable marker is represented by ctg, coding for an already partially characterized peach aux/iaa protein. its expression was shown to increase at early s most likely under a developmental control, thereafter decreasing when ethylene climacteric is fully installed. accordingly, ethylene treatments were shown to reduce the specific transcripts. besides fully agreeing with previous data, the expression profiles shown here may also represent correlative evidence for a putative functional role. indeed, no rise of expression was measured in the mesocarp of slr, consistent with the block/slowdown of development and ripening. moreover, in the case of springcrest, a high ethylene-producing variety, the rise in expression of ctg is both anticipated, paralleling ripening kinetics, and less pronounced than in fantasia, in agreement with a negative effect exerted by higher levels of ethylene. possible mechanisms involved in seed-pericarp cross-talk should take into account the vascular and cellular connections existing between the two organs. it has been shown that all the maternal tissues of pericarp and seed are intensively interconnected, while nucellar tissue is excluded from the plasmodesmata network. this implies that the flux of metabolites, as well as signaling molecules between embryo and fruit, must occur through the apoplast. taking into account that hormones play a pivotal role in the regulation of seed and fruit development, it has been assumed that they might also be involved in the cross-talk between the two organs. the heat map data will therefore be discussed taking into account the consistency of the colors in the following main two-by-two comparisons: st/h, srg/h, tfs/h, and stfs/srg. more specifically, considering the first one, consistency of colors may indicate a relationship between the hormone-related response and activation of the corresponding signal transduction pathway. in the second comparison, the same parameter may provide information about the hormone specificity of the transcriptional response, and, at the same time, of the possible cross-talk between hormones. a double comparison may allow it to be pointed out if other players besides the tfs are involved in the regulation of the downstream processes, and if a specific response is mediated by hormone-specific tfs. auxin, cytokinins, and gibberellins are generally considered to be the most relevant hormones for early seed and fruit development, whereas abscisic acid and ethylene play important roles in seed maturation and fruit ripening. from the point of view of the cross-talk between seed and mesocarp, comparisons should refer to the same developmental stage, i.e. es/em and ls/lm. concerning auxin, the data presented here point out that the specificity of the response to the hormone is higher in es and lm, although the relationship between the overall hormonometer and st data indicates that mesocarp is always more sensitive than the seed to the hormone. taking into account that the presence of a viable seed is required for fruit set and development in peach, and that the overexpression of auxin biosynthetic genes in the ovary stimulates the parthenocarpic fruit development in several species, it may be hypothesized that the signal produced by the developing seed might be either the auxin itself exported to the fruit, as demonstrated in tomato, or a secondary messenger whose target at the fruit level includes a large subset of auxin-responsive genes. this is consistent with both the high specificity of the auxin response shown here in the early developing seed and the higher sensitivity to the hormone displayed by the mesocarp paralleled by a strong hormone response. among the mesocarp auxin responsive genes, several encode elements regulating transport, indicating that auxin movement in this tissue is a relevant process, thus strengthening the hypothesis that auxin produced by the seed may behave as a signal efficiently transported to and within the mesocarp. an aux/iaa-encoding gene showed an opposite transcription profile in the two organs, being abundant in es and lm. it has been demonstrated that its tomato orthologue acts as a repressor of auxin signaling. thus, its expression in young organs seems to confirm that the hormonal response is not at the synthesis site. finally, the expression of ctg a saur-like iaa responsive gene was found to be higher in mesocarp than in seed, thus suggesting a higher auxin level in em than in es. the main process regulated by cks is cell division, occurring at early development in both seed and mesocarp. in the former, there is an up-regulation of signal transduction elements, such as ctg coding for a histidine-containing phosphotransfer protein whose transcription is abundant in very young organs. the corresponding substantial activation of hormonal targets, including several ck-specific genes, might differ in the two organs. for example, a cellulose synthase is activated in em but not in es, cytokinesis being an ls event, whereas cyclin d was up-regulated in both organs at the early stage. however, this transcriptional response did not just involve ck-specific tfs, implying that other regulatory elements may determine the hormone-specific gene activation. a similar activation of signal transduction elements to that found in the seed is present in the mesocarp at early development. however, the overall and the ck-specific target activation are not correlated to the hormone action, suggesting that cks may regulate mesocarp cell division at the post-transcriptional level, either alone or in cooperation with other phytohormones. moreover, considering the inter-organ comparison, it is noteworthy that during early development the seed displayed a higher sensitivity to cks than the mesocarp but a lower specificity of response. the amount of the overall transcriptional response observed in the seed may be due to the involvement of other hormones besides cks. during late development, an inverse situation was observed compared to the early phases. in fact, the high activation of signal transduction pathways occurring in the seed was uncoupled from the overall transcriptional response, which was even more specific in the mesocarp. the ck-mediated up-regulation of genes encoding sorbitol dehydrogenases appears particularly interesting, as this might increase the sink strength of the seed and attract photoassimilates to the entire fruit, which become more competitive in the partitioning process. from a physiological point of view, gas play a stimulatory role in fruit development, as shown by the ability to induce parthenocarpy in several species when applied in post-bloom phase and/or early development. the initial phases of endosperm and embryo development are usually related to a high level of gas, while seed maturation is paralleled by a decay of free gas and increase of their conjugates. the hormonometer data confirmed these results both in seed and mesocarp, except for tfs in the latter. in fact, the most relevant transcriptional response occurred during early development at seed level as pointed out by the es-specific expression of ctg encoding an orthologue of the arabidopsis ltp, which is classified as a ga-responsive gene involved in embryo patterning. in the mesocarp, a low correlation was observed between the tf-related transcriptional response and ga action, implying the activation of complex regulatory mechanisms that may play relevant roles in the cross-talk between seed and mesocarp. a possible mode of interaction might be the em specific expression of a gene coding for a zinc finger protein, whose arabidopsis orthologue interacts with della proteins, is repressed by ga, and causes aba accumulation when over-expressed. however, since this transcriptional response lacked specificity, it might be hypothesized that ga action also depends on the interaction with other hormones. it has recently been demonstrated that auxin induced parthenocarpy via gas in unpollinated tomato ovaries. furthermore, the peculiar expression profile of ctg encoding a gast-like protein, orthologue of arabidopsis gasa, in em is confined to s and s stages, when cell enlargement is slow. these data are in agreement with the observed inhibition of cell elongation conferred on both arabidopsis seedling and strawberry fruit over-expressing the fragaria orthologue fagast. during late development, in spite of the slight correlation existing in terms of ga-specific response, the other gene sets appeared not to be correlated to the hormone action. it may be deduced from this that the role of ga in the cross-talk between seed and mesocarp is negligible during late development. aba is known to play an antagonistic role with respect to auxin, gas and cks, as observed during fruit development in avocado and tomato. according to the hormonometer, this antagonism was largely confirmed in the seed, the transcriptional response being correlated with higher levels of the hormone in ls compared to es, also when the aba-specific subset was considered. in fact, during late seed development, aba levels are known to increase and ga-related genes such as ctg encoding a ltp-like, are down-regulated. this physiological parameter is paralleled by a consistent transcriptional response in which tfs belonging to wrky, hd, aux/iaa, bzip and dreb-like ap families are involved. given this interpretation and taking into account that during both early and late development aba st pathways and aba-target responses are more active in the mesocarp, the hormone may play a more relevant role in the development of each organ, rather than in seed-mesocarp cross-talk. in this context, the aba pool of maternal and zygotic origin may trigger independent transduction pathways. the well-known role of ethylene in peach ripening was confirmed by the higher level of transcription of its st elements measured at the mesocarp level during late development. it is worth noting that ethylene-related transcriptional response in the lm/em comparison resembles that of aba, most likely because of the significant number of transcriptional targets shared by the two hormones. this was not observed in the inter-organ assessments, in which, differently from aba, a weaker overall transcriptional response was pointed out, with the only exception being ethylene-specific tfs that were more represented in mesocarp. as regards cross-talk, the role of ethylene might be limited to the very early phase of fruit development, as demonstrated in tomato. this might be a consequence of the fact that ethylene acts mainly within the cell where it is synthesized. also in this case, the hormone of maternal and zygotic origin may activate independent pathways controlling different processes.  in this research, genes were identified marking different developmental phases of seed and mesocarp. the reliability of these molecular markers was tested in two subsequent years and a further functional validation was carried out in three different genotypes. in the latter case, data indicate that, while seed markers represent reliable tools in all the tested varieties, in the case of the mesocarp the different developmental and ripening traits of the various genotypes somewhat affect the expression of marker genes, consistently with their putative functions and cv characteristics. the most critical phases, from the point of view of mesocarp marker retrieval, were s2ii and s this might be related to the high divergence in pericarp development among the different genotypes, as pointed out above. however, this limitation may be partially overcome by using mesocarp markers as a whole, therefore increasing their discriminating power. as regards the cross-talk between seed and pericarp, possible candidate signals were identified among hormones. in the early phases, when the cross-talk is more vital for fruit set, the candidates are auxin, cks, and gas, acting either directly or indirectly as signals, whereas aba and ethylene appear to be involved later on. further investigations relying upon the availability of whole genome platforms will allow enrichment of the marker genes repertoire and elucidation of the cross-talk mechanisms between the two organs, taking into account, besides hormones, other players such as hormone peptides and micrornas. 
in fact, attempts to stimulate parthenocarpic fruit development by means of external tools have failed. as there is limited genetic and molecular information about seed-pericarp cross-talk and development in peach, a massive gene approach based on the use of the μpeach array platform and quantitative real time rt-pcr was used to study this process. further investigations relying upon the availability of whole genome platforms will allow the enrichment of a marker genes repertoire and the elucidation of players other than hormones that are involved in seed-pericarp cross-talk. results indicated that auxin, cytokinins, and gibberellins are good candidates, acting either directly or indirectly as signals during early development, when the cross-talk is more active and vital for fruit set, whereas abscisic acid and ethylene may be involved later on. among differentially expressed genes some were validated as markers in two subsequent years and in three different genotypes. mesocarp markers were a rd22-like protein, a serin-carboxypeptidase, a senescence related protein and an aux/iaa, for s s s and s respectively. field observations and a few physiological studies have demonstrated that peach embryogenesis and fruit development are tightly coupled. later in embryo development, the interactions between seed and fruit development become less strict. the microarray data, analyzed by using the hormonometer platform, allowed the identification of hormone-responsive genes, some of them putatively involved in seed-pericarp crosstalk. moreover, physiological disturbances during early embryo development lead to seed abortion and fruitlet abscission. regarding the cross-talk between seed and pericarp, possible candidate signals were identified among hormones. in this research, genes were identified marking different phases of seed and mesocarp development. seed markers were a ltp, a pr protein, a prunin and lea protein, for s s s and s respectively. the selected genes behaved as good seed markers, while for mesocarp their reliability appeared to be dependent upon developmental and ripening traits.
 field observations and a few physiological studies have demonstrated that peach embryogenesis and fruit development are tightly coupled. in fact, attempts to stimulate parthenocarpic fruit development by means of external tools have failed. moreover, physiological disturbances during early embryo development lead to seed abortion and fruitlet abscission. later in embryo development, the interactions between seed and fruit development become less strict. as there is limited genetic and molecular information about seed-pericarp cross-talk and development in peach, a massive gene approach based on the use of the μpeach array platform and quantitative real time rt-pcr was used to study this process.  a comparative analysis of the transcription profiles conducted in seed and mesocarp throughout different developmental stages evidenced that genes are differentially expressed in seed and fruit. among differentially expressed genes some were validated as markers in two subsequent years and in three different genotypes. seed markers were a ltp, a pr protein, a prunin and lea protein, for s s s and s respectively. mesocarp markers were a rd22-like protein, a serin-carboxypeptidase, a senescence related protein and an aux/iaa, for s s s and s respectively. the microarray data, analyzed by using the hormonometer platform, allowed the identification of hormone-responsive genes, some of them putatively involved in seed-pericarp crosstalk. results indicated that auxin, cytokinins, and gibberellins are good candidates, acting either directly or indirectly as signals during early development, when the cross-talk is more active and vital for fruit set, whereas abscisic acid and ethylene may be involved later on.  in this research, genes were identified marking different phases of seed and mesocarp development. the selected genes behaved as good seed markers, while for mesocarp their reliability appeared to be dependent upon developmental and ripening traits. regarding the cross-talk between seed and pericarp, possible candidate signals were identified among hormones. further investigations relying upon the availability of whole genome platforms will allow the enrichment of a marker genes repertoire and the elucidation of players other than hormones that are involved in seed-pericarp cross-talk. 