BACKGROUND
the transition from maturation to ripening in fleshy fruits can be either dependent on the hormone ethylene or not.
in the first case fruit, such as peaches, tomatoes, bananas and apples exhibit a characteristic respiratory rise and are defined climacteric, in the second case do not and are classified as non-climacteric .
it is known that climacteric fruit can produce ethylene by either a system- <dig> or a system- <dig> biosynthesis, with the latter active when autocatalytic ethylene is produced  <cit> .
system- <dig> ethylene has been shown to modulate the expression of hundreds of genes both in tomato  <cit>  and in peach  <cit> .
all plant tissues are able to produce ethylene and the gaseous hormone is involved in many developmental processes  <cit>  and in response to both biotic  <cit>  and abiotic stresses  <cit> .
in the model plant arabidopsis there are nine 1-aminocyclopropane-1-carboxylic acid  synthase  and five acc oxidase  genes, coding for different isoforms of the two enzymes involved in the conversion of s-adenosyl-methionine  to ethylene.
the unique and overlapping roles of the different members of the arabidopsis acs family have been investigated both at molecular  <cit>  and biochemical  <cit>  levels.
in the tomato genome, the model plant for fleshy fruit ripening, eleven acs and seven aco putative genes were identified, of which leacs1a, leacs <dig>  leacs <dig>  leacs <dig>  leaco <dig>  leaco <dig> and leaco <dig> are differentially expressed during ripening .
a possible auxin promoting effect on system- <dig> ethylene production in tomato fruit has not been considered in the model explaining the transition from system- <dig> to system- <dig> ethylene biosynthesis  <cit> , even though the inductive effect of auxin on acs transcription in vegetative tissues has long since been known  <cit> .
the induction of leacs <dig> by auxin, even in tomato plants with down-regulated expression of the dr <dig> gene, coding for an auxin responsive factor , has been shown to occur also in maturing fruit  <cit> .
nevertheless, auxin induction of ethylene synthesis in ripening fruit did not draw much attention, presumably because auxin has normally been considered to counteract ripening .
in peach a transcriptomic approach has highlighted a previously underestimated role of auxin in the regulation of fruit ripening  <cit> .
the requirement of auxin to switch to system- <dig> ethylene production in fruit was later shown to be the reason of the stony-hard phenotype, as fruit from this genotype was found to be unable of rising iaa concentration  <cit> .
however, being the auxin-ethylene relationship very intricate, several overlapping effects are still to be assigned to either one or the other of the two hormones.
the synthetic compound 1-methylcyclopropene  is structurally related to ethylene and widely used on many species to block its unwanted effects, as in fruit ripening and in cut flowers  <cit> .
it has been shown that 1-mcp interacts with both etr <dig> and ers <dig> proteins, thus stabilizing their repressor activity  <cit> , and for such a reason this chemical is commercially used to delay hormone’s unwanted effects.
as system- <dig> ethylene synthesis is autocatalytic, 1-mcp should block it, and this is what has been reported in many fruit, such as apple, tomato and banana .
in peach there are contrasting reports: some researchers state that 1-mcp can block ethylene synthesis, and thus delay fruit ripening  <cit> , although not efficiently  <cit> , while others found enhanced ethylene production .
by using a non destructive spectroscopic index  which can be used to asses the exact maturation and ripening phase of peach fruits  <cit>  also in stony-hard genotypes  <cit> , we could perform 1-mcp and auxin treatments on homogeneously ripe fruits.
the possibility of sorting fruits in a precise series of ripening stages has made it possible to gain new findings on the regulation of this transition by auxin and ethylene and on 1-mcp action in peach.
more interestingly, this experimental system resulted to be suitable to shed new light on the regulation of ethylene synthesis and its cross-talk with auxin, possibly mediated and/or enhanced by a peptide hormone belonging to the rgf/glv  family.
RESULTS
effect of 1-mcp on fruit ripening
in order to perform 1-mcp treatments on fruit at a homogeneous stage of ripening, the index of absorbance difference,  was used to group melting flesh peaches according to their maturity and ripening stage.
the efficacy of 1-mcp in delaying peach ripening was determined by evaluating ethylene production and flesh firmness .
as fruits belonging to class  <dig> and  <dig> were already producing ethylene, treatments were performed with both  <dig> and 5 μl l− <dig> of 1-mcp  or with 5 μl , to saturate all possible hormone binding sites.
1-mcp effect was different depending on the class.
in class  <dig> 1-mcp was effective in both reducing ethylene production  and delaying softening .
in class  <dig> 1-mcp effect was intermediate; indeed, the inhibitor speeded up ethylene production  but was able to delay fruit softening .
the experiment was stopped after 84 h because of fruit decay.
in class  <dig> 1-mcp induced ethylene production  and was ineffective on fruit softening .
the experiment was stopped after 60 h because of fruit decay.fig.
<dig> flesh firmness  and ethylene production  during post-harvest of peaches either treated  or not  with 1-mcp .
the y scale is the same in the three panels for ff , while it differs for ethylene production .
iad was used to group s <dig> fruit according to their ripening stages: class  <dig> ), class  <dig> ), and class  <dig> ).
the arrow at the bottom indicates the end of the 1-mcp treatment in 1-mcp-exposed fruit.
thereafter, fruit were kept in air at 25 °c.
data represent the mean  ± s.d
effect of 1-mcp on gene transcription
the effects of 1-mcp on the peach fruit transcriptome were evaluated by a microarray approach using the μpeach <dig>  platform  <cit> .
class  <dig> fruit kept in air for 24 h after the 1-mcp treatment  were used because they showed the highest retention of ff compared to control fruit.
a direct comparison approach  was employed.
setting the false discovery rate  to 5 %,  <dig> probes resulted to be differentially expressed .
these data are partially overlapping to those obtained with the same μpeach <dig>  platform  <cit> .
1-mcp effect on genes regulated by ripening and ethylene
microarray data were crossed with those already available on the regulation of peach ripening and exogenous ethylene application  <cit> ; this analysis highlighted:i)  <dig> probes induced by both ripening and ethylene and, as expected, repressed by 1-mcp.
these included genes encoding an endopolygalacturonase , a pyruvate decarboxylase  and a nine-cis-epoxycarotenoid dioxygenase , whose expression profiles was confirmed by quantitative reverse transcriptase real-time pcr .
ii)  <dig> probes that were down-regulated by both ripening and ethylene but up-regulated by the 1-mcp treatment.
among them were genes encoding a plasma membrane intrinsic protein , a sorbitol transporter  and a rd22-like protein , whose expression profile was confirmed by qrt-pcr .
noteworthy is that there were not genes induced by ripening, ethylene and 1-mcp nor repressed by the same conditions.
1-mcp effect on genes regulated by ripening and auxin
as done for ethylene, microarray data were crossed with those already available on the regulation of peach ripening by auxin  <cit> ; this analysis highlighted:i)  <dig> probes induced by both ripening and auxin and repressed by 1-mcp.
all these  <dig> probes fell within the group of those  <dig> induced by ripening and ethylene and repressed by 1-mcp seen above, thus confirming that their auxin responsiveness was mediated by ethylene.
ii)  <dig> probes behaved in the opposite way, that is, they were down-regulated by both ripening and auxin but up-regulated by the 1-mcp treatment.
of these,  <dig> were in common with the  <dig> probes down-regulated by ripening and ethylene and up-regulated by 1-mcp, thus confirming that also for these genes their auxin responsiveness was mediated by ethylene.
noteworthy is that microarray analysis highlighted only one gene as induced by ripening, auxin and 1-mcp  and also only one gene as repressed in the three situations .
this unexpected expression profile was confirmed by qrt-pcr for both ctg <dig> and ctg <dig> .fig.
<dig> relative expression profiles of selected genes in leaf, flower and fruit at different stages of development , in fruit at s3ii following ethylene  and naa treatment  and in preclimacteric s <dig> fruits belonging to class  <dig>  or class  <dig>  treated with 1-mcp .
genes belonging to the ethylene domain , auxin domain , transcription factors  or with the unexpected transcriptional response following 1-mcp treatment are grouped.
genes belonging to the same family are boxed.
expression values, determined by qrt-pcr, were related to the highest expression of each gene  within each experiment .
ppa no.
indicate the peach gene identifier as described in  <cit> , while ctg name indicate the cdna identifiers on the microarray μpeach <dig>  as described in  <cit> .
hormone treatments  lasted for 48 h .
srg fruits were collected at commercial maturity date and sampled after 36 h of storage either in air or in 1-mcp  plus air
regulation of system- <dig> ethylene biosynthesis
the increase in system- <dig> ethylene production measured in 1-mcp treated fruit of class  <dig> and  <dig> led us to investigate the regulation of hormone metabolism during the transition from developing to ripening fruits.
to better understand the function of the considered genes, their expression was evaluated, by means of qrt-pcr experiments, in fruits at different developmental stages and in non-fruit tissues such as leaf and flower; furthermore, their responsiveness to exogenous ethylene and 1-naphthalene acetic acid  was evaluated at the pre-climacteric stage .
transcriptional regulation of ethylene biosynthetic genes
beside the three known acs genes  <cit> , probes for five additional members of this family were designed based on est searches and the recently released peach genome sequence  <cit> .
a comparison with arabidopsis acs genes allowed us to assign acs <dig>  and acs <dig>  to group a  <cit> , and acs <dig> , acs <dig> , acs <dig>  and acs <dig>  to group b. furthermore, acs <dig>  and acs <dig>  clustered with arabidopsis atacs <dig> and atacs <dig>  and thus most likely are aminotransferases that do not act on branched chain amino acids and do not have acc synthase activity  <cit> .
therefore, they were not considered further.
the expression of acs <dig>  if any, was below the detection limit in the tested samples.
as previously described  <cit> , acs <dig>  transcription was dramatically induced by ripening .
in pre-climacteric s3ii peaches naa was much more effective than ethylene in increasing acs <dig> mrna abundance .
blocking ethylene perception with 1-mcp seemed ineffective on acs <dig> accumulation in class  <dig> fruits, while acs <dig> was strongly induced in class  <dig> fruits .
acs <dig>  expression was relatively abundant only in fully developed leaves, but it was very low in fruit, with a peak at the beginning of development .
acs <dig> mrna was almost undetectable in s3ii and s4i fruits, thus ethylene, naa and 1-mcp responsiveness could not be assessed .
acs <dig> mrna  was detected only in flowers and leaves , and, although peaking in the former, it was only a fraction of acs <dig> and acs <dig> expression .
acs <dig> was expressed at extremely low levels  in flowers and very young fruits .
in ripening fruits its expression was hardly detectable, also after treatments with ethylene, naa and 1-mcp .
acs <dig> expression was also very low and detectable only in s <dig> and s <dig> fruit, with a maximum in s4ii .
naa had a positive effect on acs <dig> mrna accumulation  as 1-mcp had on class  <dig> fruit .
as regards the acc oxidases , the well-known ripening and ethylene induced expression of aco <dig>  as well as its repression by 1-mcp  <cit>  was confirmed .
aco <dig> transcription’s dependency on ethylene was strengthened by the fact that in 1-mcp-treated fruits belonging to both class  <dig> and  <dig> there was a marked reduction of its mrna .
aco <dig> expression was almost constitutive in the tested samples with a minimum in young  fruit .
its steady state level was lower than that of aco <dig> in all tested tissues, even in developing and maturing fruits, where aco <dig> expression was at its minimum .
ethylene and, to a lesser extent, also naa, slightly induced aco <dig> transcription in pre-climacteric s3ii fruit .
surprisingly, a clear inductive effect of the 1-mcp treatment on aco <dig> expression was observed in class  <dig> and, although to a lesser extent, also class  <dig> fruit .
besides the two known ones, three additional aco genes were found in the peach genome and were named aco <dig> , aco <dig>  and aco <dig> .
aco <dig> is a truncated inactive and untranscribed version of aco <dig>  separated from it by less than  <dig> kilobases .
among the peach acos, aco <dig> was the less expressed one in tested samples .
it had a maximum in overripe fruit  and at s3ii it was strongly induced by naa .
given that its expression was very low and did not vary very much between control and treated samples, its responsiveness to 1-mcp, if any, was difficult to interpret .
expression of aco <dig> was highest at s <dig> and then decreases to be almost undetectable at ripening .
thus the slight variations observed after hormone treatments at s3ii  and after 1-mcp application  were considered of limited physiological relevance.
transcriptional regulation of ethylene receptor genes
the developmental and hormonal  control on the transcription of three known ethylene receptors was already known  <cit> .
here extensive search of the genome sequence allowed us to isolate only a fourth receptor, which was named etr <dig> .
as for the other receptor genes, also etr <dig> transcription raised with the progression of ripening to peak at s <dig> and decreased thereafter .
as for etr <dig> and ers <dig>  neither ethylene nor naa had a great impact on etr <dig> transcription, while etr <dig> mrna abundance increased after naa and, mostly, ethylene treatment .
1-mcp had almost no effect on etr <dig>  it slightly down-regulated ers <dig> and etr <dig>  while it strongly suppressed etr <dig> transcription in both class  <dig> and class  <dig> fruit , thus confirming previous findings  <cit> .
transcriptional regulation of genes belonging to the auxin domain
to further investigate the relationship between ethylene and auxin during peach fruit ripening, the expression of several genes belonging to the auxin domain was evaluated.
of the aux/iaa genes shown to be up-regulated during peach ripening , five were induced by the ethylene inhibitor .
interestingly, of these five genes, only three  were strongly induced by naa at s3ii , with the latter strongly up-regulated also by ethylene.
in addition, the transcription of two tir <dig> auxin receptors  was abundant at ripening .
less clear was their ethylene and auxin responsiveness, as both genes were repressed by the hormones at s3ii  and mildly regulated by 1-mcp .
ctg <dig> was induced while ctg <dig> response depended on the class .
a similar behavior was observed also for the ripening specific  and ethylene induced  pin <dig>  gene , thus confirming that class  <dig> and class  <dig> fruits behave differently  <cit> .
application of 1-mcp was almost ineffective on genes involved in auxin biosynthesis such as tryptophan synthase beta subunit , and indole-3-glycerol phosphate synthase , that were induced at ripening  <cit> .
on the contrary, it was very effective in inducing the transcription of three previously uncharacterized genes , two of which belong to the auxin domain.
two genes whose products are involved in maintaining auxin homeostasis had a transcriptional profile almost overlapping with that of ctg <dig>  in particular, ctg <dig> codes for an iaa amidohydrolase highly similar to arabidopsis iaa-leucine resistant  <dig>  and its abundance sharply increased during climacteric ripening .
this gene was positively regulated by naa and insensitive to ethylene ; furthermore, it was stimulated by 1-mcp in both class  <dig> and  <dig> fruit .
the second gene  codes for a gh <dig> protein, an iaa-amido synthase, and it was expressed almost exclusively during fruit ripening ; its transcription was induced by naa in pre-climacteric s3ii fruit  and by 1-mcp, especially in class <dig> fruit .
transcriptional regulation of ripening-related transcription factors
given the known importance of the role on ripening of transcription factors  belonging to different families, the expression of five genes, whose orthologs have been characterized in other systems  <cit> , was tested.
a sepallata-like mads-box , which is highly similar to tomato rin  <cit> , had the highest expression in s4ii fruits , was induced by both ethylene and naa at s3ii , and seemed to be slightly repressed by 1-mcp in class  <dig> fruit .
similarly, a nam tf , sharing strong similarity to tomato nor  <cit> , accumulated in mesocarp during ripening to peak at the end of the process , was induced by both ethylene and naa at s3ii , and seemed repressed by 1-mcp .
also two hormone-related tfs, the first mediating auxin  and the second ethylene  responses, had a ripening-related expression , but while the first was negatively regulated by both hormones at s3ii, the latter was induced, especially by naa .
the unusual hormonal regulation of this erf was confirmed by the 1-mcp treatment, which was ineffective on its expression, while the arf responded differently in the two classes .
expression, structure, homology and putative function of ctg134
the gene  corresponding to ctg <dig> was the only one to be highlighted by microarray analyses as induced at the s3ii to s4i transition and by naa and 1-mcp.
this peculiar transcription profile was confirmed by qrt-pcr, which revealed that, besides in class  <dig>  also in class  <dig> fruit 1-mcp induced its mrna abundance .
moreover, the mrna abundance of ctg <dig> was strongly increased by naa and repressed by ethylene in pre-climacteric s3ii fruit .
in tissues other than ripening fruit at s <dig>  ctg <dig> mrna was hardly detectable .
the mrna corresponding to ctg <dig> codes for a protein of  <dig> aa with a predicted molecular mass of  <dig>  kda.
this polypeptide shares very low similarity with other plant proteins but for a small sequence of  <dig> amino acids  at its carboxy terminus .
like many other signaling peptides, this short hydrophilic protein has a predicted n-terminal sequence  of about 23– <dig> aa that most likely directs it to the secretory pathway.
the mature, apoplastic protein is rich in charged residues  and, although different in sequence, its structure resembles that of signaling peptides of the rgf/glv type  <cit> .
the c-ter peptide sequence is highly conserved in a number of recently characterized arabidopsis proteins .fig.
<dig> structure of the ctg <dig> protein.
hydrophobicity plot of the protein sequence predicted from ctg <dig>  and amino acid alignment of the c-ter with the corresponding part of some arabidopsis rgfs/glvs.
the mature peptide hormone  is released from the mature protein  after delivery in the cell wall
1-mcp increases free auxin levels in peach ripening fruits
as the transcription of several ripening- and iaa-induced genes was induced in 1-mcp-treated peaches, auxin was quantified in the same samples used for the rna expression data of fig.
2c and in class  <dig> fruit at harvest .
the iaa concentration was lowest in class  <dig> fruit, reached a maximum in class  <dig> and slightly decreased thereafter .
on the contrary, ethylene levels were hardly detectable in class  <dig> fruit, slightly increased in class  <dig> and peaked in class  <dig>  thus showing that the auxin peak preceded that of ethylene .
also abscisic acid , long since known to accumulate in mesocarp of peach ripening fruits  <cit> , and recently claimed to be among the determinants of ripening of several climacteric fruits  <cit>  including peach  <cit> , gradually increased from class  <dig> to class  <dig> fruit .fig.
<dig> auxin, ethylene  and aba levels during fruit ripening ) and following 1-mcp treatment , aba in panel ).
srg peaches were sampled after 36 h of storage either in air or in 1-mcp  plus air .
bars are the standard deviations from the means of three or more replicates.
letters above columns indicate significant differences with a tuckey hsd test at p < <dig>
when the effect of 1-mcp on the iaa concentration was considered, it was clear that the ethylene inhibitor induced the amount of auxin in both class  <dig> and class  <dig> fruit .
it has to be noted that, at the same time point , 1-mcp did not alter ethylene production, but only its action .
blocked ethylene perception did not significantly alter aba concentration in class  <dig>  while it reduced it in class  <dig> fruits .
timing and hierarchy of the hormonal signals during ripening
to better clarify timing and hierarchy of the hormonal cascade that leads to climacteric ripening , the s3ii-s4i transition in melting flesh redhaven  peaches was investigated with a better temporal resolution than that of fig.
2a, that spanned whole fruit development .
also in this case, fruits, collected on the same day, were correctly graded by means of their iad values into six classes .
furthermore, fruits from a selection carrying the “stony hard” trait , known for its inability to produce ethylene during ripening  <cit> , were used and also grouped according to their ida values .
a subset of the genes used in fig.
<dig> were selected as exemplificative of their groups  and the absolute quantification of their transcripts determined in the nine samples.
in this experiment, the absolute mrna abundance was determined to allow precise comparison between rxx and rh and, within the same genotype, among genes of the same families .
of the genes involved in ethylene synthesis in rh, acs <dig> showed the strongest transcriptional repression in rxx fruits .
in rh, its ripening-induced expression started earlier than that of aco <dig>  whose expression, together with those of the other acos, was not significantly repressed in rxx fruit .
among the genes of the iaa domain, it was the irl1-like ctg <dig> mrna that peaked in class  <dig> fruit, immediately before acs <dig> rise.
also iaa perception was critical in class 0/ <dig> fruit, as evidenced by the expression of tir1/ctg <dig>  which, it has to be noted, was very similar to that of the ethylene receptors etr <dig> and etr <dig>  however, while receptors and iaa biosynthesis genes were expressed at comparable amounts also in rxx fruit, this did not occur for ilr1-like ctg <dig>  nor for gh <dig>  and iaa <dig> , whose products are involved in iaa catabolism and signal transduction, respectively, and were induced by iaa.
on the contrary, the expression of etr <dig>  etr <dig> and etr <dig> in rxx was similar, if not higher, to that found in rh.
in addition, the expression of ripening related transcription factors showed the cruciality of class  <dig>  stage, that we propose to be at the turning point of system- <dig> to system- <dig> ethylene synthesis.
moreover, the fact that the expression of the tfs is similar in the two genotypes supports that the stony hard trait is not due to alteration in their transcription, as it is for cnr in tomato  <cit> .
striking expression differences were found for ctg <dig>  which was almost undetectable in rxx fruits.
the nced <dig> mrnas gradually accumulated in rh fruits as ripening proceeded, while their levels in rxx were comparable to those found in class −1/ <dig> in rh.
lastly, the cell wall genes confirmed many previous reports on their different transcriptional regulation, with pg expression strictly dependent on ethylene, while exp <dig> transcription, albeit peaking before the climacteric  and being repressed by both ethylene and auxin  <cit> , also needed a fruit in a ripening status that is incomplete in rxx.fig.
<dig> absolute expression profiles of selected genes at the transition from maturation to ripening in melting flesh  and stony-hard  genotypes.
gene groups and colors are as for fig.
<dig> but for the last column .
as quantification was carried out with a standard, comparison of the relative abundance among members of the same gene family has been added
different competences to auxin in preclimacteric fruit
the effect of auxin on ethylene synthesis was tested on three classes of rh fruits .
on class − <dig> fruits , the synthetic auxin naa had an inhibitory effect on ethylene synthesis .
on the contrary, on class  <dig> and class  <dig> fruits auxin had a positive effect on ethylene production, being the induction stronger in class  <dig> after 12 h while the amplitude more pronounced on class  <dig> after 60 h from the treatment .
also class  <dig> fruits of the rxx genotype were able to produce ethylene after the naa treatment, although the total amount of the hormone produced was much lower than that of the climacteric genotype , thus confirming recent findings  <cit> .fig.
<dig> effect of auxin treatment on the ability to produce ethylene in fruit at different ripening stages in melting flesh –, respectively) and stony-hard  genotypes.
c: control, fruits treated with a mock solution; naa: fruits treated with a solution containing naa .
bars are the standard deviations from the means of three or more replicates
the different behavior of class − <dig> compared to class  <dig> and class  <dig> fruit in rh was confirmed also at the transcriptional level .
indeed, both acs <dig> and ctg <dig> were repressed 36 h after the treatment in class − <dig>  while they showed an opposite trend  in class  <dig> fruit .
this opposite behavior was detected also in other auxin-inducible genes, as gh <dig>  while ethylene regulated genes as aco <dig> and etr <dig> showed a marked up-regulation at both time-points in class  <dig> fruit , in agreement with the measured ethylene production .
ethylene biosynthetic genes acs <dig> and aco <dig> were more expressed in naa treated rxx fruit .
also etr <dig> was more expressed 36 h after the treatment, while etr <dig> was not.
the effectiveness of the naa treatment was visible also on gh <dig> and, albeit at a lower extent, also on ilr <dig> gene expression, which were both induced, specially at 12 h, while expression of clb/ctg <dig>  which normally decreases during ripening, was higher in controls than in treated samples, meaning that the latter were riper.
nced <dig> expression was induced by naa both in rxx  and class  <dig> rh  fruits, but not in class − <dig>  pg confirmed its strong dependency to ethylene for its expression, being repressed in class − <dig> and induced in class  <dig> rh fruits .
the positive impact of naa on ethylene synthesis in rxx fruits allowed a transient induction of pg expression .
on the contrary, the pre-climacteric ethylene-independent expression of exp <dig> was confirmed in rh fruits .
the only gene whose expression was almost undetectable in rxx fruits also after the naa treatment was ctg <dig> .fig.
<dig> relative expression profiles of selected genes following auxin treatment in fruit at different ripening stages in melting flesh  and stony-hard  genotypes.
gene groups and colors are as for fig.
2
dna sequence of ctg <dig> in the rxx genotype
the expression of ctg <dig> was absent in the stony hard genotype.
for this reason, its sequence in this genotype was determined starting at 2782 bp before the atg start codon down to 512 bp after the stop codon.
the analyzed region did not contain any structural variation nor any polymorphism, thus being identical to the reference genome  <cit> .
discussion
effect of 1-mcp on peach fruit ripening
the efficacy of 1-mcp in delaying peach fruit ripening is controversial.
there are reports that both support an inhibitory action  <cit>  and others, which state that the chemical is  ineffective .
here we showed that its effects are largely dependent on the ripening stage at which the chemical is applied.
we were able to make such a distinction due to the use of the non destructive index of absorbance difference , which can estimate the fruit ripening stage by means of a computer-assisted spectrophotometric device  <cit> .
thus, if 1-mcp was supplied at an early ripening stage , a gross parameter such as pulp softening supports the chemical efficacy in delaying fruit ripening.
on the other hand, the chemical was ineffective in delaying softening of class  <dig> fruits, thus indicating that the maturity stage of application is critical.
contrary to what happens in other fruit such as apple  <cit> , pear  <cit> , tomato  <cit>  but also in stone fruit as plum  <cit> , in peach 1-mcp did not inhibit ethylene biosynthesis in class  <dig> and  <dig> fruits , thus confirming previous results  <cit>  but it did in class  <dig> fruits, where it was also effective in delaying softening .
thus, two apparently contradictory effects, which were seen at their best in class  <dig> fruits , were due to 1-mcp application on peaches: the delay of fruit softening, i.e.
of the ethylene response, and the stimulation of ethylene production.
softening delay was efficient only when the ethylene evolution was low, probably because genes encoding cell wall degrading enzymes were induced with very low amount of hormone, as this has been shown for the tomato pg  <cit> .
this finding might explain the contradictory reports on the effect of 1-mcp on both ethylene production and ripening delay in peach fruit present in the literature .
albeit not being useful as a post-harvest tool for the peach industry, the biological effect of 1-mcp was confirmed at the molecular level by transcriptome changes that the chemical could cause.
many  1-mcp inhibited genes were also ripening and ethylene induced, thus confirming previous findings  <cit>  about the importance of the hormone during peach ripening.
on the contrary, 1-mcp had a positive effect on many ripening and ethylene repressed genes , confirming its ability to delay the progression of the syndrome over a short time.
regulation of system- <dig> ethylene biosynthesis
system- <dig> ethylene production is largely dependent on the expression of acs <dig>  <cit>  and aco <dig>  <cit> .
the expression of other members of the two families  does not fit with the model of the transition from system- <dig> to system- <dig> proposed in tomato  <cit>  and apple  <cit> .
of the four newly described putative acs genes  only two  can be considered bona fide true acss, while acs <dig> and acs <dig>  being closely related to atacs <dig> and atacs <dig>  most likely lack acs activity  <cit> .
all acs mrnas but acs <dig> were almost undetectable during fruit ripening.
acs <dig> and acs <dig>  expressed in flower, could be involved in the ethylene production occurring during pollination  <cit>  or organ shedding  <cit> .
the unwanted wounding in the field might be the reason for the expression of the wound-inducible acs <dig>  <cit>  in fully expanded leaves.
acs <dig> and acs <dig> are expressed in fruits at early stages and thus it is possible that, together with acs <dig>  they are responsible for ethylene production in young fruits.
nonetheless, it is conceivable to exclude that they have a role similar to tomato leacs <dig>  <cit>  or apple mdacs <dig>  <cit>  that, being expressed during the transition from system- <dig> to system- <dig>  allow to rise ethylene concentration over the threshold necessary to start its autocatalytic production.
regulation of ethylene perception
during the initial phase of the transition from system- <dig> to system- <dig>  a crucial role in sensing ethylene might be carried out by etr <dig> and ers <dig> whose expression increased at ripening but did not seem to be controlled by either ethylene or auxin, so that they could be considered under developmental control  <cit> .
only the expression of etr <dig> seemed to be associated with ripening in an ethylene-dependent manner.
indeed, when ethylene was not sensed, as in the presence of 1-mcp, etr <dig> transcription was strongly inhibited.
in the peach genome sequence  <cit>  only an additional ethylene receptor  was isolated, thus bringing to only four the members of this family in peach, while there are five in arabidopsis  <cit>  and seven in tomato  <cit> .
nonetheless, etr <dig> extremely low expression seemed to exclude a main role for it in ripening.
auxin homeostasis
the effect of auxin on ethylene production has not only been demonstrated in vivo but also in vitro.
the ethylene amount produced by mesocarp disks cultured in vitro depends on both the concentration of naa in the medium and the age of the fruits used to prepare the explants  <cit> .
in particular, ethylene production was higher and faster if disks were obtained from fruits near ripening treated with 100 μm naa.
the increase of free auxin  <cit>  during the last stages of peach ripening might not rely only on de-novo synthesis but also on its release from conjugated forms.
indeed, the expression of ctg <dig> , coding for an iaa amidohydrolase of the ilr <dig>  type, involved in the release of iaa from iaa-leu  correlates with free iaa content during ripening  <cit>  and following 1-mcp treatment  better than that of genes involved in auxin biosynthesis .
however, we can not exclude that genes similar to arabidopsis taa <dig>  <cit>  and yuccas  <cit>  also allow rapid and direct iaa synthesis from tryptophan also in peach and this will be investigated in the near future.
to guarantee the correct auxin homeostasis, the expression of ctg <dig> seemed to be counterbalanced by that of ctg <dig>  coding for a gh <dig> protein.
gh <dig> proteins have been demonstrated to be iaa-amido synthases which help to maintain auxin homeostasis by conjugating excess iaa to amino acids  <cit> .
both the genes encoding the iaa amidohydrolase  and the gh <dig>  protein were strongly induced by naa and expressed almost exclusively in s <dig> fruit.
furthermore, their expression was extremely low in the rxx genotype but it could be rescued by exogenous auxin .
this peculiar expression profile is very similar to that of acs <dig> and the expression of these three genes might be the cause of the peaks of both auxin and ethylene measured in s <dig> fruit  <cit> .
however, transcript profiling on more transition stages from pre-climacteric to climacteric fruits  together with hormone quantifications  allowed us to find that auxin peaked before ethylene production increased.
1-mcp induction of auxin-induced genes
the microarray experiment pointed out the peculiar regulation of ctg <dig> and ctg <dig>  encoding a peptide hormone and a calcineurin b-like protein, respectively.
the regulation of these genes was unexpected since most of those that are both ripening and auxin-induced are also 1-mcp-repressed while those that are ripening and auxin-repressed are also 1-mcp-induced.
this is true for those genes whose auxin-regulation is indirect, because it is ethylene-mediated .
however in peach there are also ripening-regulated genes directly responding to auxin  <cit> .
besides ctg <dig>  lowering the stringency of the selection parameters pointed out that other genes belonging to the auxin domain and that were naa-induced were 1-mcp-induced too.
the expression of some of these genes is often considered diagnostic for increased level of auxin in the tissue from which the rna has been obtained.
the expression profiles of genes such as those coding for gh <dig>  and aux/iaa proteins  suggested that a rise in free auxin concentration had probably occurred in peaches following the 1-mcp treatment.
auxin measurements in fruits confirmed this hypothesis  and, based on gene expression data, we propose that this increase is, at least partly, mediated by the activity of the amidohydrolase encoded by the ctg <dig> gene.
the 1-mcp effects on ethylene synthesis depended on the physiological state of the peach fruits .
in addition, the kinetic of the induction of transcription of genes belonging to the auxin domain were different in class  <dig> and class  <dig> fruit.
genes like ctg <dig> and ctg <dig> were strongly induced both in class  <dig> and in class  <dig> fruit, while others, such as ctg <dig> better responded in class  <dig> fruit.
the different physiological status of fruits belonging to different ida classes were confirmed also in rxx and rh peaches.
it was clarified that auxin has an inductive effect on ripening only if a given maturation state is reached, otherwise it inhibits the process.
the fact that blocking ethylene perception stimulated the auxin synthesis needed for the system- <dig> ethylene production tells about the importance of an intact receptor apparatus also in peach, as it has been shown by inverse genetics in tomato  <cit> .
these differences could be appreciated only because the fruit sorting was finely tuned due to the use of the iad index  <cit>  and probably are the results of a cascade of signals leading to fruit ripening.
interactions with other hormones
the increase of aba content during peach ripening has long since been known and considered to be dependent on ethylene  <cit> .
the idea of a possible ethylene control on aba synthesis is here strengthened by the fact that nced <dig> , but also ctg <dig> , was strongly inhibited by 1-mcp, besides being induced by ripening, ethylene and naa.
however it has also been reported that aba reach its height before the ethylene peak  <cit>  and recently it has been shown that, if applied when competence to ripening has been acquired, aba has an inductive effect, besides on ripening, also on the regulation of ethylene biosynthetic and auxin responsive genes  <cit> .
this extended hormone cross-talk could have even a wider relevance since it has been observed also in roots of cleavers.
in the latter system nced expression was directly induced by iaa and sustained by ethylene, so that both contributed to an induced aba synthesis  <cit> .
in peach it could be that ethylene and aba control each other to synchronize different aspects of ripening.
a model for the transition of ethylene biosynthesis from system- <dig> to system- <dig> in peach
there are several scenarios that might be hypothesized in which the actors and their interaction that lead to the transition from system- <dig> to system- <dig> ethylene production might be placed.
based on expression profiles of ripening related genes and their responsiveness to ethylene, 1-mcp and naa in melting flesh  and stony hard  genotypes, we propose that the transition is initiated, together with other not yet characterized developmental signals such as the activity of tfs  <cit> , by the increase in free auxin levels, possibly at a stage similar to class  <dig> in rh .
acs <dig> transcription, that might depend also on rin-like mads, as observed in tomato  <cit> , is the rate limiting step of system- <dig> ethylene synthesis, and can rely only on auxin increase but not on other acs genes expression to switch ethylene synthesis from system- <dig> to system- <dig>  since ethylene production must be controlled, a possible feed-back regulation involves the hormone sensing carried out by etr <dig>  ers <dig>  etr <dig> and possibly etr <dig>  the newly synthesized ethylene negatively regulates the mrna abundance of amidohydrolase ctg <dig> but not of gh <dig> ctg <dig>  thus allowing a balanced auxin homeostasis.
when ethylene concentration is low  or receptors are blocked by 1-mcp, fruit cells that have completed their maturation sense that ethylene is missing, so its synthesis has to be induced by a release of free auxin.
the ctg <dig> gene has several features that make it a good candidate as a possible component of the rheostat that balances auxin and ethylene synthesis.
its naa induced expression is almost exclusively limited to fruit at early s <dig> and inhibited by ethylene.
and when ethylene should be there  but it is not sensed because of the presence of 1-mcp, ctg <dig> transcription raises very quickly, especially in fruit at the stage in which there is the transition from system- <dig> to system- <dig> .
the regulation of the expression of the peptide encoded by polaris  in arabidopsis  and its involvement in a regulatory loop of auxin–ethylene interactions indicates that the cross-talk between ethylene and auxin can be mediated by signaling peptide  <cit> .
the  <dig> aa sequence at the c-terminus of the ctg <dig> protein is highly conserved with arabidopsis rgf/glv like peptides  <cit> ; thus, besides being involved in root meristem maintenance  <cit>  and in root gravitropic response  <cit>  rgf/glv peptides are involved also in fruit ripening.
the ability of those peptides to control auxin distribution and to reinforce its action by regulating the turnover of an auxin efflux carrier  <cit> , thus regulating auxin gradients, might explain the ethylene induced expression of ctg <dig>  coding for a peach pin <dig>  as ripening usually commences in several regions of a fruit, the predicted apoplastic localization of ctg <dig>  together with ethylene diffusion, might help the spreading of ripening from cell to cell throughout the fruit.
altogether these data lead to the hypothesis that the transition from system- <dig> to system- <dig> ethylene biosynthesis in peach fruit is controlled by a regulatory loop of auxin-ethylene interactions in which hormone levels are reciprocally controlled by a signaling system involving a rgf/glv peptide hormone.
CONCLUSIONS
here we showed that blocking ethylene receptors with 1-mcp increases free auxin content in ripening peach fruit, thus leading to ethylene overproduction.
this increase is sustained by the transcriptional activation of ilr1-like ctg <dig> and thus, at least partly, by auxin de-conjugation.
the ctg <dig> protein, a precursor of a peptide hormone of the rgf/glv type is a good candidate to mediate this ethylene-auxin cross-talk.
the auxin-dependent rise in ethylene concentration represses many of the auxin genes, among which also ilr1-like ctg <dig>  and that coding for ctg <dig>  this new player opens the theoretical possibility to design new rational and environmentally friendly agrochemicals useful to control ripening in those crops, as peach, where 1-mcp is ineffective and cold storage has many drawbacks.

 the transition from maturation to ripening in fleshy fruits can be either dependent on the hormone ethylene or not. in the first case fruit, such as peaches, tomatoes, bananas and apples exhibit a characteristic respiratory rise and are defined climacteric, in the second case do not and are classified as non-climacteric. it is known that climacteric fruit can produce ethylene by either a system- or a system- biosynthesis, with the latter active when autocatalytic ethylene is produced. system- ethylene has been shown to modulate the expression of hundreds of genes both in tomato and in peach. all plant tissues are able to produce ethylene and the gaseous hormone is involved in many developmental processes and in response to both biotic and abiotic stresses. in the model plant arabidopsis there are nine 1-aminocyclopropane-1-carboxylic acid synthase and five acc oxidase genes, coding for different isoforms of the two enzymes involved in the conversion of s-adenosyl-methionine to ethylene. the unique and overlapping roles of the different members of the arabidopsis acs family have been investigated both at molecular and biochemical levels. in the tomato genome, the model plant for fleshy fruit ripening, eleven acs and seven aco putative genes were identified, of which leacs1a, leacs leacs leacs leaco leaco and leaco are differentially expressed during ripening. a possible auxin promoting effect on system- ethylene production in tomato fruit has not been considered in the model explaining the transition from system- to system- ethylene biosynthesis, even though the inductive effect of auxin on acs transcription in vegetative tissues has long since been known. the induction of leacs by auxin, even in tomato plants with down-regulated expression of the dr gene, coding for an auxin responsive factor, has been shown to occur also in maturing fruit. nevertheless, auxin induction of ethylene synthesis in ripening fruit did not draw much attention, presumably because auxin has normally been considered to counteract ripening. in peach a transcriptomic approach has highlighted a previously underestimated role of auxin in the regulation of fruit ripening. the requirement of auxin to switch to system- ethylene production in fruit was later shown to be the reason of the stony-hard phenotype, as fruit from this genotype was found to be unable of rising iaa concentration. however, being the auxin-ethylene relationship very intricate, several overlapping effects are still to be assigned to either one or the other of the two hormones. the synthetic compound 1-methylcyclopropene is structurally related to ethylene and widely used on many species to block its unwanted effects, as in fruit ripening and in cut flowers. it has been shown that 1-mcp interacts with both etr and ers proteins, thus stabilizing their repressor activity, and for such a reason this chemical is commercially used to delay hormone’s unwanted effects. as system- ethylene synthesis is autocatalytic, 1-mcp should block it, and this is what has been reported in many fruit, such as apple, tomato and banana. in peach there are contrasting reports: some researchers state that 1-mcp can block ethylene synthesis, and thus delay fruit ripening, although not efficiently, while others found enhanced ethylene production. by using a non destructive spectroscopic index which can be used to asses the exact maturation and ripening phase of peach fruits also in stony-hard genotypes, we could perform 1-mcp and auxin treatments on homogeneously ripe fruits. the possibility of sorting fruits in a precise series of ripening stages has made it possible to gain new findings on the regulation of this transition by auxin and ethylene and on 1-mcp action in peach. more interestingly, this experimental system resulted to be suitable to shed new light on the regulation of ethylene synthesis and its cross-talk with auxin, possibly mediated and/or enhanced by a peptide hormone belonging to the rgf/glv family.  effect of 1-mcp on fruit ripening in order to perform 1-mcp treatments on fruit at a homogeneous stage of ripening, the index of absorbance difference, was used to group melting flesh peaches according to their maturity and ripening stage. the efficacy of 1-mcp in delaying peach ripening was determined by evaluating ethylene production and flesh firmness. as fruits belonging to class and were already producing ethylene, treatments were performed with both and 5 μl l− of 1-mcp or with 5 μl, to saturate all possible hormone binding sites. 1-mcp effect was different depending on the class. in class 1-mcp was effective in both reducing ethylene production and delaying softening. in class 1-mcp effect was intermediate; indeed, the inhibitor speeded up ethylene production but was able to delay fruit softening. the experiment was stopped after 84 h because of fruit decay. in class 1-mcp induced ethylene production and was ineffective on fruit softening. the experiment was stopped after 60 h because of fruit decay.fig. flesh firmness and ethylene production during post-harvest of peaches either treated or not with 1-mcp. the y scale is the same in the three panels for ff, while it differs for ethylene production. iad was used to group s fruit according to their ripening stages: class ), class ), and class ). the arrow at the bottom indicates the end of the 1-mcp treatment in 1-mcp-exposed fruit. thereafter, fruit were kept in air at 25 °c. data represent the mean  ± s.d effect of 1-mcp on gene transcription the effects of 1-mcp on the peach fruit transcriptome were evaluated by a microarray approach using the μpeach platform. class fruit kept in air for 24 h after the 1-mcp treatment were used because they showed the highest retention of ff compared to control fruit. a direct comparison approach was employed. setting the false discovery rate to 5 %, probes resulted to be differentially expressed. these data are partially overlapping to those obtained with the same μpeach platform. 1-mcp effect on genes regulated by ripening and ethylene microarray data were crossed with those already available on the regulation of peach ripening and exogenous ethylene application; this analysis highlighted:i) probes induced by both ripening and ethylene and, as expected, repressed by 1-mcp. these included genes encoding an endopolygalacturonase, a pyruvate decarboxylase and a nine-cis-epoxycarotenoid dioxygenase, whose expression profiles was confirmed by quantitative reverse transcriptase real-time pcr. ii) probes that were down-regulated by both ripening and ethylene but up-regulated by the 1-mcp treatment. among them were genes encoding a plasma membrane intrinsic protein, a sorbitol transporter and a rd22-like protein, whose expression profile was confirmed by qrt-pcr. noteworthy is that there were not genes induced by ripening, ethylene and 1-mcp nor repressed by the same conditions. 1-mcp effect on genes regulated by ripening and auxin as done for ethylene, microarray data were crossed with those already available on the regulation of peach ripening by auxin; this analysis highlighted:i) probes induced by both ripening and auxin and repressed by 1-mcp. all these probes fell within the group of those induced by ripening and ethylene and repressed by 1-mcp seen above, thus confirming that their auxin responsiveness was mediated by ethylene. ii) probes behaved in the opposite way, that is, they were down-regulated by both ripening and auxin but up-regulated by the 1-mcp treatment. of these, were in common with the probes down-regulated by ripening and ethylene and up-regulated by 1-mcp, thus confirming that also for these genes their auxin responsiveness was mediated by ethylene. noteworthy is that microarray analysis highlighted only one gene as induced by ripening, auxin and 1-mcp and also only one gene as repressed in the three situations. this unexpected expression profile was confirmed by qrt-pcr for both ctg and ctg.fig. relative expression profiles of selected genes in leaf, flower and fruit at different stages of development, in fruit at s3ii following ethylene and naa treatment and in preclimacteric s fruits belonging to class or class treated with 1-mcp. genes belonging to the ethylene domain, auxin domain, transcription factors or with the unexpected transcriptional response following 1-mcp treatment are grouped. genes belonging to the same family are boxed. expression values, determined by qrt-pcr, were related to the highest expression of each gene within each experiment. ppa no. indicate the peach gene identifier as described in, while ctg name indicate the cdna identifiers on the microarray μpeach as described in. hormone treatments lasted for 48 h. srg fruits were collected at commercial maturity date and sampled after 36 h of storage either in air or in 1-mcp plus air regulation of system- ethylene biosynthesis the increase in system- ethylene production measured in 1-mcp treated fruit of class and led us to investigate the regulation of hormone metabolism during the transition from developing to ripening fruits. to better understand the function of the considered genes, their expression was evaluated, by means of qrt-pcr experiments, in fruits at different developmental stages and in non-fruit tissues such as leaf and flower; furthermore, their responsiveness to exogenous ethylene and 1-naphthalene acetic acid was evaluated at the pre-climacteric stage. transcriptional regulation of ethylene biosynthetic genes beside the three known acs genes, probes for five additional members of this family were designed based on est searches and the recently released peach genome sequence. a comparison with arabidopsis acs genes allowed us to assign acs and acs to group a, and acs, acs, acs and acs to group b. furthermore, acs and acs clustered with arabidopsis atacs and atacs and thus most likely are aminotransferases that do not act on branched chain amino acids and do not have acc synthase activity. therefore, they were not considered further. the expression of acs if any, was below the detection limit in the tested samples. as previously described, acs transcription was dramatically induced by ripening. in pre-climacteric s3ii peaches naa was much more effective than ethylene in increasing acs mrna abundance. blocking ethylene perception with 1-mcp seemed ineffective on acs accumulation in class fruits, while acs was strongly induced in class fruits. acs expression was relatively abundant only in fully developed leaves, but it was very low in fruit, with a peak at the beginning of development. acs mrna was almost undetectable in s3ii and s4i fruits, thus ethylene, naa and 1-mcp responsiveness could not be assessed. acs mrna was detected only in flowers and leaves, and, although peaking in the former, it was only a fraction of acs and acs expression. acs was expressed at extremely low levels in flowers and very young fruits. in ripening fruits its expression was hardly detectable, also after treatments with ethylene, naa and 1-mcp. acs expression was also very low and detectable only in s and s fruit, with a maximum in s4ii. naa had a positive effect on acs mrna accumulation as 1-mcp had on class fruit. as regards the acc oxidases, the well-known ripening and ethylene induced expression of aco as well as its repression by 1-mcp was confirmed. aco transcription’s dependency on ethylene was strengthened by the fact that in 1-mcp-treated fruits belonging to both class and there was a marked reduction of its mrna. aco expression was almost constitutive in the tested samples with a minimum in young fruit. its steady state level was lower than that of aco in all tested tissues, even in developing and maturing fruits, where aco expression was at its minimum. ethylene and, to a lesser extent, also naa, slightly induced aco transcription in pre-climacteric s3ii fruit. surprisingly, a clear inductive effect of the 1-mcp treatment on aco expression was observed in class and, although to a lesser extent, also class fruit. besides the two known ones, three additional aco genes were found in the peach genome and were named aco, aco and aco. aco is a truncated inactive and untranscribed version of aco separated from it by less than kilobases. among the peach acos, aco was the less expressed one in tested samples. it had a maximum in overripe fruit and at s3ii it was strongly induced by naa. given that its expression was very low and did not vary very much between control and treated samples, its responsiveness to 1-mcp, if any, was difficult to interpret. expression of aco was highest at s and then decreases to be almost undetectable at ripening. thus the slight variations observed after hormone treatments at s3ii and after 1-mcp application were considered of limited physiological relevance. transcriptional regulation of ethylene receptor genes the developmental and hormonal control on the transcription of three known ethylene receptors was already known. here extensive search of the genome sequence allowed us to isolate only a fourth receptor, which was named etr. as for the other receptor genes, also etr transcription raised with the progression of ripening to peak at s and decreased thereafter. as for etr and ers neither ethylene nor naa had a great impact on etr transcription, while etr mrna abundance increased after naa and, mostly, ethylene treatment. 1-mcp had almost no effect on etr it slightly down-regulated ers and etr while it strongly suppressed etr transcription in both class and class fruit, thus confirming previous findings. transcriptional regulation of genes belonging to the auxin domain to further investigate the relationship between ethylene and auxin during peach fruit ripening, the expression of several genes belonging to the auxin domain was evaluated. of the aux/iaa genes shown to be up-regulated during peach ripening, five were induced by the ethylene inhibitor. interestingly, of these five genes, only three were strongly induced by naa at s3ii, with the latter strongly up-regulated also by ethylene. in addition, the transcription of two tir auxin receptors was abundant at ripening. less clear was their ethylene and auxin responsiveness, as both genes were repressed by the hormones at s3ii and mildly regulated by 1-mcp. ctg was induced while ctg response depended on the class. a similar behavior was observed also for the ripening specific and ethylene induced pin gene, thus confirming that class and class fruits behave differently. application of 1-mcp was almost ineffective on genes involved in auxin biosynthesis such as tryptophan synthase beta subunit, and indole-3-glycerol phosphate synthase, that were induced at ripening. on the contrary, it was very effective in inducing the transcription of three previously uncharacterized genes, two of which belong to the auxin domain. two genes whose products are involved in maintaining auxin homeostasis had a transcriptional profile almost overlapping with that of ctg in particular, ctg codes for an iaa amidohydrolase highly similar to arabidopsis iaa-leucine resistant and its abundance sharply increased during climacteric ripening. this gene was positively regulated by naa and insensitive to ethylene; furthermore, it was stimulated by 1-mcp in both class and fruit. the second gene codes for a gh protein, an iaa-amido synthase, and it was expressed almost exclusively during fruit ripening; its transcription was induced by naa in pre-climacteric s3ii fruit and by 1-mcp, especially in class fruit. transcriptional regulation of ripening-related transcription factors given the known importance of the role on ripening of transcription factors belonging to different families, the expression of five genes, whose orthologs have been characterized in other systems, was tested. a sepallata-like mads-box, which is highly similar to tomato rin, had the highest expression in s4ii fruits, was induced by both ethylene and naa at s3ii, and seemed to be slightly repressed by 1-mcp in class fruit. similarly, a nam tf, sharing strong similarity to tomato nor, accumulated in mesocarp during ripening to peak at the end of the process, was induced by both ethylene and naa at s3ii, and seemed repressed by 1-mcp. also two hormone-related tfs, the first mediating auxin and the second ethylene responses, had a ripening-related expression, but while the first was negatively regulated by both hormones at s3ii, the latter was induced, especially by naa. the unusual hormonal regulation of this erf was confirmed by the 1-mcp treatment, which was ineffective on its expression, while the arf responded differently in the two classes. expression, structure, homology and putative function of ctg134 the gene corresponding to ctg was the only one to be highlighted by microarray analyses as induced at the s3ii to s4i transition and by naa and 1-mcp. this peculiar transcription profile was confirmed by qrt-pcr, which revealed that, besides in class also in class fruit 1-mcp induced its mrna abundance. moreover, the mrna abundance of ctg was strongly increased by naa and repressed by ethylene in pre-climacteric s3ii fruit. in tissues other than ripening fruit at s ctg mrna was hardly detectable. the mrna corresponding to ctg codes for a protein of aa with a predicted molecular mass of  kda. this polypeptide shares very low similarity with other plant proteins but for a small sequence of amino acids at its carboxy terminus. like many other signaling peptides, this short hydrophilic protein has a predicted n-terminal sequence of about 23– aa that most likely directs it to the secretory pathway. the mature, apoplastic protein is rich in charged residues and, although different in sequence, its structure resembles that of signaling peptides of the rgf/glv type. the c-ter peptide sequence is highly conserved in a number of recently characterized arabidopsis proteins.fig. structure of the ctg protein. hydrophobicity plot of the protein sequence predicted from ctg and amino acid alignment of the c-ter with the corresponding part of some arabidopsis rgfs/glvs. the mature peptide hormone is released from the mature protein after delivery in the cell wall 1-mcp increases free auxin levels in peach ripening fruits as the transcription of several ripening- and iaa-induced genes was induced in 1-mcp-treated peaches, auxin was quantified in the same samples used for the rna expression data of fig. 2c and in class fruit at harvest. the iaa concentration was lowest in class fruit, reached a maximum in class and slightly decreased thereafter. on the contrary, ethylene levels were hardly detectable in class fruit, slightly increased in class and peaked in class thus showing that the auxin peak preceded that of ethylene. also abscisic acid, long since known to accumulate in mesocarp of peach ripening fruits, and recently claimed to be among the determinants of ripening of several climacteric fruits including peach, gradually increased from class to class fruit.fig. auxin, ethylene and aba levels during fruit ripening ) and following 1-mcp treatment, aba in panel ). srg peaches were sampled after 36 h of storage either in air or in 1-mcp plus air. bars are the standard deviations from the means of three or more replicates. letters above columns indicate significant differences with a tuckey hsd test at p < when the effect of 1-mcp on the iaa concentration was considered, it was clear that the ethylene inhibitor induced the amount of auxin in both class and class fruit. it has to be noted that, at the same time point, 1-mcp did not alter ethylene production, but only its action. blocked ethylene perception did not significantly alter aba concentration in class while it reduced it in class fruits. timing and hierarchy of the hormonal signals during ripening to better clarify timing and hierarchy of the hormonal cascade that leads to climacteric ripening, the s3ii-s4i transition in melting flesh redhaven peaches was investigated with a better temporal resolution than that of fig. 2a, that spanned whole fruit development. also in this case, fruits, collected on the same day, were correctly graded by means of their iad values into six classes. furthermore, fruits from a selection carrying the “stony hard” trait, known for its inability to produce ethylene during ripening, were used and also grouped according to their ida values. a subset of the genes used in fig.  were selected as exemplificative of their groups and the absolute quantification of their transcripts determined in the nine samples. in this experiment, the absolute mrna abundance was determined to allow precise comparison between rxx and rh and, within the same genotype, among genes of the same families. of the genes involved in ethylene synthesis in rh, acs showed the strongest transcriptional repression in rxx fruits. in rh, its ripening-induced expression started earlier than that of aco whose expression, together with those of the other acos, was not significantly repressed in rxx fruit. among the genes of the iaa domain, it was the irl1-like ctg mrna that peaked in class fruit, immediately before acs rise. also iaa perception was critical in class 0/ fruit, as evidenced by the expression of tir1/ctg which, it has to be noted, was very similar to that of the ethylene receptors etr and etr however, while receptors and iaa biosynthesis genes were expressed at comparable amounts also in rxx fruit, this did not occur for ilr1-like ctg nor for gh and iaa, whose products are involved in iaa catabolism and signal transduction, respectively, and were induced by iaa. on the contrary, the expression of etr etr and etr in rxx was similar, if not higher, to that found in rh. in addition, the expression of ripening related transcription factors showed the cruciality of class stage, that we propose to be at the turning point of system- to system- ethylene synthesis. moreover, the fact that the expression of the tfs is similar in the two genotypes supports that the stony hard trait is not due to alteration in their transcription, as it is for cnr in tomato. striking expression differences were found for ctg which was almost undetectable in rxx fruits. the nced mrnas gradually accumulated in rh fruits as ripening proceeded, while their levels in rxx were comparable to those found in class −1/ in rh. lastly, the cell wall genes confirmed many previous reports on their different transcriptional regulation, with pg expression strictly dependent on ethylene, while exp transcription, albeit peaking before the climacteric and being repressed by both ethylene and auxin, also needed a fruit in a ripening status that is incomplete in rxx.fig. absolute expression profiles of selected genes at the transition from maturation to ripening in melting flesh and stony-hard genotypes. gene groups and colors are as for fig.  but for the last column. as quantification was carried out with a standard, comparison of the relative abundance among members of the same gene family has been added different competences to auxin in preclimacteric fruit the effect of auxin on ethylene synthesis was tested on three classes of rh fruits. on class − fruits, the synthetic auxin naa had an inhibitory effect on ethylene synthesis. on the contrary, on class and class fruits auxin had a positive effect on ethylene production, being the induction stronger in class after 12 h while the amplitude more pronounced on class after 60 h from the treatment. also class fruits of the rxx genotype were able to produce ethylene after the naa treatment, although the total amount of the hormone produced was much lower than that of the climacteric genotype, thus confirming recent findings.fig. effect of auxin treatment on the ability to produce ethylene in fruit at different ripening stages in melting flesh –, respectively) and stony-hard genotypes. c: control, fruits treated with a mock solution; naa: fruits treated with a solution containing naa. bars are the standard deviations from the means of three or more replicates the different behavior of class − compared to class and class fruit in rh was confirmed also at the transcriptional level. indeed, both acs and ctg were repressed 36 h after the treatment in class − while they showed an opposite trend in class fruit. this opposite behavior was detected also in other auxin-inducible genes, as gh while ethylene regulated genes as aco and etr showed a marked up-regulation at both time-points in class fruit, in agreement with the measured ethylene production. ethylene biosynthetic genes acs and aco were more expressed in naa treated rxx fruit. also etr was more expressed 36 h after the treatment, while etr was not. the effectiveness of the naa treatment was visible also on gh and, albeit at a lower extent, also on ilr gene expression, which were both induced, specially at 12 h, while expression of clb/ctg which normally decreases during ripening, was higher in controls than in treated samples, meaning that the latter were riper. nced expression was induced by naa both in rxx and class rh fruits, but not in class − pg confirmed its strong dependency to ethylene for its expression, being repressed in class − and induced in class rh fruits. the positive impact of naa on ethylene synthesis in rxx fruits allowed a transient induction of pg expression. on the contrary, the pre-climacteric ethylene-independent expression of exp was confirmed in rh fruits. the only gene whose expression was almost undetectable in rxx fruits also after the naa treatment was ctg.fig. relative expression profiles of selected genes following auxin treatment in fruit at different ripening stages in melting flesh and stony-hard genotypes. gene groups and colors are as for fig. 2 dna sequence of ctg in the rxx genotype the expression of ctg was absent in the stony hard genotype. for this reason, its sequence in this genotype was determined starting at 2782 bp before the atg start codon down to 512 bp after the stop codon. the analyzed region did not contain any structural variation nor any polymorphism, thus being identical to the reference genome. discussion effect of 1-mcp on peach fruit ripening the efficacy of 1-mcp in delaying peach fruit ripening is controversial. there are reports that both support an inhibitory action and others, which state that the chemical is ineffective. here we showed that its effects are largely dependent on the ripening stage at which the chemical is applied. we were able to make such a distinction due to the use of the non destructive index of absorbance difference, which can estimate the fruit ripening stage by means of a computer-assisted spectrophotometric device. thus, if 1-mcp was supplied at an early ripening stage, a gross parameter such as pulp softening supports the chemical efficacy in delaying fruit ripening. on the other hand, the chemical was ineffective in delaying softening of class fruits, thus indicating that the maturity stage of application is critical. contrary to what happens in other fruit such as apple, pear, tomato but also in stone fruit as plum, in peach 1-mcp did not inhibit ethylene biosynthesis in class and fruits, thus confirming previous results but it did in class fruits, where it was also effective in delaying softening. thus, two apparently contradictory effects, which were seen at their best in class fruits, were due to 1-mcp application on peaches: the delay of fruit softening, i.e. of the ethylene response, and the stimulation of ethylene production. softening delay was efficient only when the ethylene evolution was low, probably because genes encoding cell wall degrading enzymes were induced with very low amount of hormone, as this has been shown for the tomato pg. this finding might explain the contradictory reports on the effect of 1-mcp on both ethylene production and ripening delay in peach fruit present in the literature. albeit not being useful as a post-harvest tool for the peach industry, the biological effect of 1-mcp was confirmed at the molecular level by transcriptome changes that the chemical could cause. many 1-mcp inhibited genes were also ripening and ethylene induced, thus confirming previous findings about the importance of the hormone during peach ripening. on the contrary, 1-mcp had a positive effect on many ripening and ethylene repressed genes, confirming its ability to delay the progression of the syndrome over a short time. regulation of system- ethylene biosynthesis system- ethylene production is largely dependent on the expression of acs and aco. the expression of other members of the two families does not fit with the model of the transition from system- to system- proposed in tomato and apple. of the four newly described putative acs genes only two can be considered bona fide true acss, while acs and acs being closely related to atacs and atacs most likely lack acs activity. all acs mrnas but acs were almost undetectable during fruit ripening. acs and acs expressed in flower, could be involved in the ethylene production occurring during pollination or organ shedding. the unwanted wounding in the field might be the reason for the expression of the wound-inducible acs in fully expanded leaves. acs and acs are expressed in fruits at early stages and thus it is possible that, together with acs they are responsible for ethylene production in young fruits. nonetheless, it is conceivable to exclude that they have a role similar to tomato leacs or apple mdacs that, being expressed during the transition from system- to system- allow to rise ethylene concentration over the threshold necessary to start its autocatalytic production. regulation of ethylene perception during the initial phase of the transition from system- to system- a crucial role in sensing ethylene might be carried out by etr and ers whose expression increased at ripening but did not seem to be controlled by either ethylene or auxin, so that they could be considered under developmental control. only the expression of etr seemed to be associated with ripening in an ethylene-dependent manner. indeed, when ethylene was not sensed, as in the presence of 1-mcp, etr transcription was strongly inhibited. in the peach genome sequence only an additional ethylene receptor was isolated, thus bringing to only four the members of this family in peach, while there are five in arabidopsis and seven in tomato. nonetheless, etr extremely low expression seemed to exclude a main role for it in ripening. auxin homeostasis the effect of auxin on ethylene production has not only been demonstrated in vivo but also in vitro. the ethylene amount produced by mesocarp disks cultured in vitro depends on both the concentration of naa in the medium and the age of the fruits used to prepare the explants. in particular, ethylene production was higher and faster if disks were obtained from fruits near ripening treated with 100 μm naa. the increase of free auxin during the last stages of peach ripening might not rely only on de-novo synthesis but also on its release from conjugated forms. indeed, the expression of ctg, coding for an iaa amidohydrolase of the ilr type, involved in the release of iaa from iaa-leu correlates with free iaa content during ripening and following 1-mcp treatment better than that of genes involved in auxin biosynthesis. however, we can not exclude that genes similar to arabidopsis taa and yuccas also allow rapid and direct iaa synthesis from tryptophan also in peach and this will be investigated in the near future. to guarantee the correct auxin homeostasis, the expression of ctg seemed to be counterbalanced by that of ctg coding for a gh protein. gh proteins have been demonstrated to be iaa-amido synthases which help to maintain auxin homeostasis by conjugating excess iaa to amino acids. both the genes encoding the iaa amidohydrolase and the gh protein were strongly induced by naa and expressed almost exclusively in s fruit. furthermore, their expression was extremely low in the rxx genotype but it could be rescued by exogenous auxin. this peculiar expression profile is very similar to that of acs and the expression of these three genes might be the cause of the peaks of both auxin and ethylene measured in s fruit. however, transcript profiling on more transition stages from pre-climacteric to climacteric fruits together with hormone quantifications allowed us to find that auxin peaked before ethylene production increased. 1-mcp induction of auxin-induced genes the microarray experiment pointed out the peculiar regulation of ctg and ctg encoding a peptide hormone and a calcineurin b-like protein, respectively. the regulation of these genes was unexpected since most of those that are both ripening and auxin-induced are also 1-mcp-repressed while those that are ripening and auxin-repressed are also 1-mcp-induced. this is true for those genes whose auxin-regulation is indirect, because it is ethylene-mediated. however in peach there are also ripening-regulated genes directly responding to auxin. besides ctg lowering the stringency of the selection parameters pointed out that other genes belonging to the auxin domain and that were naa-induced were 1-mcp-induced too. the expression of some of these genes is often considered diagnostic for increased level of auxin in the tissue from which the rna has been obtained. the expression profiles of genes such as those coding for gh and aux/iaa proteins suggested that a rise in free auxin concentration had probably occurred in peaches following the 1-mcp treatment. auxin measurements in fruits confirmed this hypothesis and, based on gene expression data, we propose that this increase is, at least partly, mediated by the activity of the amidohydrolase encoded by the ctg gene. the 1-mcp effects on ethylene synthesis depended on the physiological state of the peach fruits. in addition, the kinetic of the induction of transcription of genes belonging to the auxin domain were different in class and class fruit. genes like ctg and ctg were strongly induced both in class and in class fruit, while others, such as ctg better responded in class fruit. the different physiological status of fruits belonging to different ida classes were confirmed also in rxx and rh peaches. it was clarified that auxin has an inductive effect on ripening only if a given maturation state is reached, otherwise it inhibits the process. the fact that blocking ethylene perception stimulated the auxin synthesis needed for the system- ethylene production tells about the importance of an intact receptor apparatus also in peach, as it has been shown by inverse genetics in tomato. these differences could be appreciated only because the fruit sorting was finely tuned due to the use of the iad index and probably are the results of a cascade of signals leading to fruit ripening. interactions with other hormones the increase of aba content during peach ripening has long since been known and considered to be dependent on ethylene. the idea of a possible ethylene control on aba synthesis is here strengthened by the fact that nced, but also ctg, was strongly inhibited by 1-mcp, besides being induced by ripening, ethylene and naa. however it has also been reported that aba reach its height before the ethylene peak and recently it has been shown that, if applied when competence to ripening has been acquired, aba has an inductive effect, besides on ripening, also on the regulation of ethylene biosynthetic and auxin responsive genes. this extended hormone cross-talk could have even a wider relevance since it has been observed also in roots of cleavers. in the latter system nced expression was directly induced by iaa and sustained by ethylene, so that both contributed to an induced aba synthesis. in peach it could be that ethylene and aba control each other to synchronize different aspects of ripening. a model for the transition of ethylene biosynthesis from system- to system- in peach there are several scenarios that might be hypothesized in which the actors and their interaction that lead to the transition from system- to system- ethylene production might be placed. based on expression profiles of ripening related genes and their responsiveness to ethylene, 1-mcp and naa in melting flesh and stony hard genotypes, we propose that the transition is initiated, together with other not yet characterized developmental signals such as the activity of tfs, by the increase in free auxin levels, possibly at a stage similar to class in rh. acs transcription, that might depend also on rin-like mads, as observed in tomato, is the rate limiting step of system- ethylene synthesis, and can rely only on auxin increase but not on other acs genes expression to switch ethylene synthesis from system- to system- since ethylene production must be controlled, a possible feed-back regulation involves the hormone sensing carried out by etr ers etr and possibly etr the newly synthesized ethylene negatively regulates the mrna abundance of amidohydrolase ctg but not of gh ctg thus allowing a balanced auxin homeostasis. when ethylene concentration is low or receptors are blocked by 1-mcp, fruit cells that have completed their maturation sense that ethylene is missing, so its synthesis has to be induced by a release of free auxin. the ctg gene has several features that make it a good candidate as a possible component of the rheostat that balances auxin and ethylene synthesis. its naa induced expression is almost exclusively limited to fruit at early s and inhibited by ethylene. and when ethylene should be there but it is not sensed because of the presence of 1-mcp, ctg transcription raises very quickly, especially in fruit at the stage in which there is the transition from system- to system-. the regulation of the expression of the peptide encoded by polaris in arabidopsis and its involvement in a regulatory loop of auxin–ethylene interactions indicates that the cross-talk between ethylene and auxin can be mediated by signaling peptide. the aa sequence at the c-terminus of the ctg protein is highly conserved with arabidopsis rgf/glv like peptides; thus, besides being involved in root meristem maintenance and in root gravitropic response rgf/glv peptides are involved also in fruit ripening. the ability of those peptides to control auxin distribution and to reinforce its action by regulating the turnover of an auxin efflux carrier, thus regulating auxin gradients, might explain the ethylene induced expression of ctg coding for a peach pin as ripening usually commences in several regions of a fruit, the predicted apoplastic localization of ctg together with ethylene diffusion, might help the spreading of ripening from cell to cell throughout the fruit. altogether these data lead to the hypothesis that the transition from system- to system- ethylene biosynthesis in peach fruit is controlled by a regulatory loop of auxin-ethylene interactions in which hormone levels are reciprocally controlled by a signaling system involving a rgf/glv peptide hormone.  here we showed that blocking ethylene receptors with 1-mcp increases free auxin content in ripening peach fruit, thus leading to ethylene overproduction. this increase is sustained by the transcriptional activation of ilr1-like ctg and thus, at least partly, by auxin de-conjugation. the ctg protein, a precursor of a peptide hormone of the rgf/glv type is a good candidate to mediate this ethylene-auxin cross-talk. the auxin-dependent rise in ethylene concentration represses many of the auxin genes, among which also ilr1-like ctg and that coding for ctg this new player opens the theoretical possibility to design new rational and environmentally friendly agrochemicals useful to control ripening in those crops, as peach, where 1-mcp is ineffective and cold storage has many drawbacks. 
as a consequence, a burst in ethylene production speeding up ripening occurs. only glv ctg expression could not be rescued by exogenous auxin. to elucidate this interaction at the transition from maturation to ripening and the accompanying switch from system- to system- ethylene biosynthesis, fruits of melting flesh and stony hard genotypes, the latter unable to produce system- ethylene because of insufficient amount of auxin at ripening, were treated with auxin, ethylene and with 1-methylcyclopropene, known to block ethylene receptors. 1-mcp treatment of peach fruit is ineffective in delaying ripening because it stimulates an increase in free auxin. in melting flesh peaches, auxin is necessary for system- ethylene synthesis and a cross-talk between ethylene and auxin occurs during the ripening process. a melting flesh and a stony hard genotype, differing for their ability to synthetize auxin and ethylene amounts at ripening, were used to study the fine temporal regulation and auxin responsiveness of genes involved in the process. transcriptional waves showed a tight interdependence between auxin and ethylene actions with the former possibly enhanced by the glv ctg the expression of genes involved in the regulation of ripening, among which are several transcription factors, was similar in the two genotypes or could be rescued by auxin application in the stony hard. the nature and transcriptional response of ctg led to discover a rise in free auxin in 1-mcp treated fruit. unexpectedly, 1-mcp induced genes also up-regulated by ripening, ethylene and auxin, as ctg similar to golven peptides, and repressed genes also down-regulated by ripening, ethylene and auxin, as ctg a calcineurin b-like protein. the effects of the treatments on the different genotypes were monitored by hormone quantifications and transcription profiling.
 in melting flesh peaches, auxin is necessary for system- ethylene synthesis and a cross-talk between ethylene and auxin occurs during the ripening process. to elucidate this interaction at the transition from maturation to ripening and the accompanying switch from system- to system- ethylene biosynthesis, fruits of melting flesh and stony hard genotypes, the latter unable to produce system- ethylene because of insufficient amount of auxin at ripening, were treated with auxin, ethylene and with 1-methylcyclopropene, known to block ethylene receptors. the effects of the treatments on the different genotypes were monitored by hormone quantifications and transcription profiling.  in melting flesh fruit, 1-mcp responses differed according to the ripening stage. unexpectedly, 1-mcp induced genes also up-regulated by ripening, ethylene and auxin, as ctg similar to golven peptides, and repressed genes also down-regulated by ripening, ethylene and auxin, as ctg a calcineurin b-like protein. the nature and transcriptional response of ctg led to discover a rise in free auxin in 1-mcp treated fruit. this increase was supported by the induced transcription of ctg an iaa-amino acid hydrolase. a melting flesh and a stony hard genotype, differing for their ability to synthetize auxin and ethylene amounts at ripening, were used to study the fine temporal regulation and auxin responsiveness of genes involved in the process. transcriptional waves showed a tight interdependence between auxin and ethylene actions with the former possibly enhanced by the glv ctg the expression of genes involved in the regulation of ripening, among which are several transcription factors, was similar in the two genotypes or could be rescued by auxin application in the stony hard. only glv ctg expression could not be rescued by exogenous auxin.  1-mcp treatment of peach fruit is ineffective in delaying ripening because it stimulates an increase in free auxin. as a consequence, a burst in ethylene production speeding up ripening occurs. based on a network of gene transcriptional regulations, a model in which appropriate level of ctg peptide hormone might be necessary to allow the correct balance between auxin and ethylene for peach ripening to occur is proposed. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords 1-methylcyclopropene index of absorbance difference microarraynectarineprunus persicahormone peptidegolvenroot growth factorissue-copyright-statement© the author 2016 