Our study differs from prior peach transcriptomic analyses in two ways. First, we are using samples from pools of genetically related siblings with contrasting sensitivity to chilling injury subjected or not to cold storage. Thus we expect to reveal genes whose expression patterns are linked to the different cold sensitivity, while leveraging transcript differences associated with other phenotypic traits, as it would be the case when comparing only two peach cultivars that have different chilling susceptibilities in addition to other phenotypic differences. Second, by medium throughput qRT PCR we extended our microarray results derived from the comparison of the contrasting pools to a relatively large number of 15 individual lines from the same population differing in the mealiness sensibility and the gene expression results of the selected genes were consistent with their individual sensitivity level.Orthologs of several transcription factors found upregulated similarly in S and LS cold-treated fruits have been previously reported as being up-regulated during cold acclimation in Arabidopsis and some of them also were described as belonging to a given cold acclimation regulon. This suggests the activation of a cold response program in peach fruits in part similar to those described for Arabidopsis cold acclimation. Despite observing similarities some genes exhibited an opposite trend compared to Arabidopsis which may partially reflect the sensitive character of peach fruit to cold . Several studies have associated cold tolerance and cold acclimation the transcriptional activation of genes encoding heat-shock proteins , chaperonins, LEA proteins, antioxidant/scavenging systems and related to protein synthesis.
Genes in these functional categories were generally down-regulated by cold storage in both LS and S fruits, square plastic planter what correlates well with their sensitivity to cold. Further, the orthologs of HSF4B and HSP21 were up-regulated peach fruits, whilst were down-regulated in Arabidopsis. This is particularly interesting as these genes are highly up-regulated in Arabidopsis chilling sensitive mutants upon chilling treatment. It should be noted that we are comparing the transcriptomes of different species and tissues at various physiological and growth stages, and it is likely that some differences in strategies to cope with exposure to low temperatures operate in each case. The basic question is: why do LS PopGG siblings tolerate better cold storage than S? Our results indicate that during cold storage fruits LS maintain higher levels of expression for a series of components of the antioxidant system, structure maintenance proteins and protein synthesis at least during the first week of storage . In addition, the orthologs of some TF with a higher expression levels in tolerant peach fruits have been reported to be upregulated by cold and/or other biotic or abiotic stresses in Arabidopsis . In this sense, our data indicated that the peach orthologs for genes in ICE1, CBF and HOS9 regulons may be implicated in the tolerance of fruits LS. The central role played by the ICE1-CBF cold response pathway in cold acclimation and cold tolerance is well-established in plants and has been demonstrated to exist in a wide range of plants, although, there are differences in the regulation or the size of their CBF regulons. The existence of ICE-CBF pathway has been also confirmed in fruits. Further, LeCBF1 expression levels correlates positively with cold tolerance in tomato fruits. We found that genes in the regulons ICE1and CBF were the most contributing to discriminate samples S from LS, and/or to separate samples that will become mealy, or not .
Moreover, PCA analysis identified CBF1 as the second gene that contribute the most to separate the S and LS series and qRT PCR analysis showed that the expression levels of CBF1 correlate well with the tolerance/ sensitivity of the individual pop-DG siblings . Thus, confirming ICE-CBF as important actors in the differential response to chilling between peaches S and LS. In the case of the genes in regulon HOS9 our results suggest that it is more likely related with the ability to up-regulate or to maintain similar expression levels to those observed in M fruits . Zhu et al. concluded that HOS9 must be important for both the constitutive expression and cold-induced expression of the genes that may be required for full tolerance to freezing stress. These results are consistent with peaches having the basic components of a cold response pathway, but additional studies will be required to elucidate their size and how they are regulated. In normal commercial fruit operations cold storage, involves also complete darkness. Gene by gene comparisons has revealed that around 3% of our cold regulated genes in peaches could be related to darkness . Moreover, we identify some genes whose orthologs have been described in the regulation or in response to light . Several, light sinaling elements among which were GI, DFL2, PHYA and FYPP3 were repressed by cold storage in both LS and S , consistently with the storage in darkness conditions. In addition, genes differentially expressed between fruits S and T include a number of regulators involved in light response that indicates we should take into account this factor as contributing the differential response observed in peach fruits. In Arabidopsis light is required for cold induction of several genes involved in cold acclimation, including CBFs and some light signaling mutants have impaired cold acclimation. Thus the differential response to cold storage of fruits S and LS probably have to do fruits’ ability to deal with cold and darkness. However, further experiments are required to determine in more detail the nature of the interaction between the cold and the darkness during storage.Despite no visible mealiness symptoms are observed during cold storage, the BSGA indicated dramatic changes in the peach transcriptome in response to the exposure to mealiness-inducing temperatures in a manner that these changes could be useful to predict future mealiness development .
We propose the transcript differences observed while in the cold might underlie the molecular basis of a mealiness phenotype which is still undetectable, but will be fully developed later during shelf life. This is in agreement with previous reports of the cold induction of specific target genes that are associated with the mealiness disorder. Surprisingly, our results showed that cell wall is not found among enriched categories in none of the clusters/comparisons performed on cold stored samples, suggesting that although specific changes in cell wall remodeling transcript are detected most of the changes would probably occur during shelf life. Our results reveal also that transport and signaling elements presented higher levels in S fruits, which in some cases, correlated well with the eventual mealiness phenotype. We found the orthologs of genes described as positive regulators of ABA signaling and/or osmotic stress and transporters related to Na+ and K+, sugar and nitrate homeostasis among genes high expressed in fruits S This suggests that fruits S during cold storage undergo some sort of dehydration or osmotic adjustment. It has been proposed that during cold storage, before mealiness is manifested, pectin depolymerisation but not deesterification is inhibited, square plastic plant pot what may lead to the formation of gel-forming pectins that traps free water from the surrounding tissue. As no significant differences in global water content are found between LS and S fruits it is likely that water is being lost from the cell to be trapped on the pectins of the cell wall, which still would be sensed as loss of internal water by the cell. Among genes with higher expression in sensitive fruits we identified components of auxin and ethylene signaling cascades as well the orthologs of genes involved in the biosynthesis of ABA, auxin and ethylene . We must highlight the large list of genes related to auxins among with were positive regulators of auxin responses and transporter locations . In addition, among the genes high expressed in the fruits LS at one week there were the orthologs of genes such as HAB1, PP2CA/AHG3, SAD1 and ERD15 ,which have all been described as negative regulators of ABA signaling, and IAA17/AUX3, proposed to be a negative regulator in auxin and ABA signaling. Ethylene and auxins has been described in the regulation of the ripening program of peach fruits and their involvement in the cold response has been described for Arabidopsis, tomato, apple and peach. Our results indicate that part of the ripening program probably continues during cold storage in sensitive fruits . Hence, we could expect that interactions between cold and hormones controlling the peach ripening program, which are differential between fruits S and T, impact the way fruits respond to cold and ripen afterwards during shelf life. Because the activity of most of these genes is mainly determined at post-trasncriptional level reviewed in [90], it is not possible from expression data only to infer the role of these genes during cold storage. However from our data it is clear that all three hormones may play a role in regulating the differential response of peach fruits to cold and they seem operate in association with dehydration/osmotic stress. In support of that, the orthologs of many of hormone related genes higher expressed in CS1-S fruits have been described previously either in relation to drought and osmotic stress . For example, the orthologs of SKIP, BRM and ERD1 mediate the responses or are induced by ABA, salinity and dehydration stress; CPL2 modulates auxin responses, plant growth and osmotic stress and EIN2 has been described to be an important cross-link node for the interaction of ethylene, ABA and plant response to abiotic stress. We cannot rule out that the ‘‘sensitivity’’ program is the consequence or the cause of low levels ICE1-CBF regulons. It is possible that the up-regulation of a set of common genes concomitantly with low CBF levels triggers this program. It is also feasible that among CS1 S.LS there are genes which negatively regulate the CBF response. To support this, EIN2 has been described as a negative regulator of plant response to freezing stress by negatively regulating the expression of CBF1-3 and its target genes; interestingly, CBF genes have been found to be directly repressed by IAA. Finally, it may also be possible that this program is activated to compensate efficient acclimation during cold storage. It has been described that hos9 mutants hyperactivate some cold-regulated genes through a compensating response to their increased cold sensitivity.At the mature stage specific differences at the gene expression level between the pools of fruits S and T already exist . Although our approach used pools of fruits in accordance to how they respond to cold storage, therefore minimizing differences in other aspects between genotypes, we can’t dismiss the possibility that these differences have nothing to do with adaptation to cold. Preformed mechanisms have been described in both biotic and abiotic stress tolerance and we previously identified a subset of genes differentially expressed at harvest that correlate well with CI. Cell wall metabolism has been extensively related to mealiness in peach fruits, and it has been reported that endopolygalacturonase plays a qualitative role in the mealiness expression. Our results indicate that the composition of the cell wall at harvest could play a role in the tolerance or sensitivity of peach fruits to withstand cold storage. This is in agreement with previous results. In addition the type of functional categories for the differentially expressed genes at the stage M, and the fact that most of these genes continue to show these differences during cold storage , suggest the possibility that a pre-programmed tolerance/sensitivity mechanism can be partly established previously to cold. Among the highly expressed genes in fruits LS at the mature stage, we found orthologs of genes such as CHS/TT4 and GST12/TT19 , which have been described being essential for anthocyanin and proanthocyanin accumulation. Anthocyanins have been related with browning in peaches. However, no significant differences in browning, bleeding nor in ppLDOX expression were observed between our pools. It is suggested that AtTT19 functions as a carrier to transport proanthocyanin precursors to the tonoplast to be later secreted and linked to cell wall polysaccharides. Binding that depends on the composition of the proanthocyanin. The tt19 mutation leads to the formation of aberrant PA derivatives. Thus is possible that differences in TT19 have to do with cell wall composition and chilling sensitivity. Further experiments are required to test this hypothesis. In addition, flavonoids act as negative regulators of auxin transport.