Understanding of the regulatory network involved in vegetative growth cessation and dormancy induction is still limited [1, 2, 32]. We used SSH PCR to identify differentially expressed genes in apical tissue between WT peach and the dormancy-incapable evg mutant.
We found 17 significantly up-regulated genes in the WT with respect to the mutant. Interestingly, more than 25% of the genes could not have a putative function assigned. A similar proportion of unclassifiable genes were reported in previous studies of dormancy in woody species indicating that representation of seasonally expressed genes in existing databases is low .
When considering the WT expression changes following transfer from LD to SD, three patterns could be defined. A first group of genes showed expression only during two weeks after transfer to SD. A second group of genes showed increased expression since the first week after transfer to SD and that was maintained steady or then was similar to values before the transfer. The third group of genes showed progressively enhanced expression throughout all weeks, and especially following growth cessation (weeks 4 to 8). Two major phases of gene expression response to SD were found previously in poplar: an early response to SD during the first two weeks and then a late adaptation . In another study in poplar, gene expression changed after about three weeks of SD, when bud scales were visible, and after this point there was a large reduction in the number of expressed genes and their expression level .
An interesting case of early response is the ABP20 gene whose expression peaked coincident with growth cessation and decreased 10-fold after terminal meristems were unable to resume growth (weeks 4 and 8). The peach ABP20 is related to germin and germin-like genes, which belong to the ancient superfamily of cupin proteins. The ABP20 contains a region which shared 40% of amino acid identity with a putative auxin binding site in ABP1, an auxin-binding protein isolated from maize coleoptiles . This region of homology corresponds with a BoxA domain, whose structure has been suggested to be conserved among proteins that have auxin binding-activity [33, 34]. The localization of ABP20 in the cell wall and its ability to produce H2O2 suggest a similar biological function to germin, which is related with expansion and lignification of the cell wall . The ABP1 protein of Arabidopsis has been also associated with the auxin-induced cell elongation  and has been found to be essential for the auxin control of the cell cycle using tobacco cell culture . Recent studies support the hypothesis of an auxin extracellular receptor role for ABP1 [38, 39]. ABP20 gene expression throughout the development of peach vegetative buds was previously reported . In a recent proteomic analysis, the ABP20 protein content in peach bark tissue decreased in after 5 weeks of SD treatment . Several genes involved in auxin metabolism and transport were found down-regulated in the same tissue type and conditions . It has been observed that auxin levels do not change in cambial cells during the dormancy period, but the responsiveness to auxin does [1, 41]. Although not definitive, it is tempting to speculate that there may be a role for ABP20 protein in the process of growth cessation in bud tissue by modulating the perception of auxin. However, this hypothesis will have to be specifically tested.
Another early responding gene is the putative amidase. Differential expression of the amidase gene could correspond with the different rate of growth between WT and evg genotypes, due to the core metabolic function of amidase proteins, however, a specific signalling role cannot be dismissed.
The putative LIM and KEG genes are two cases of steady response with up-regulation during the first week after transfer to SD with this elevated expression maintained similar after that point. Functional analysis is lacking for the peach putative LIM. The LIM protein gene family participates in processes such as gene transcription, cellular organization and signalling . Their essential roles have been well characterized in animals; however, only a few members have been studied in plants . A better characterized protein is KEG, a protein capable of mediating ubiquitylation. In Arabidopsis, KEG has an essential role in ABA signalling. During post-germination development, KEG protein is found in Arabidopsis seedlings . The model proposed for KEG function is the ubiquitylation and subsequent degradation of ABI5 (ABSCISIC ACID-INSENSITIVE5) and ABI3 by KEG in the absence of ABA, thus decreasing their ability to suppress growth. In the presence of ABA, this degradation is slowed to allow the transduction cascades resulting in a suppression of growth . There are commonalities between bud and seed dormancy, and although the inducing mechanism might not be shared directly, similar signalling circuits could be adopted .
Other steady responding genes are the putative GH18 family gene and unknown1. The GH18 subfamily includes chitinases with diverse defence-related functions. Some of them do not have chitinase activity , although the putative glycoside hydrolase found in this work exhibited a conserved motif that dictates enzymatic activity. Its expression was found to be up-regulated in WT. GH18 transcripts were found preferentially in active rather than dormant poplar buds . Several chitinases associated with defence-related functions have been found to be up-regulated in Populus dormant cambium tissue and peach bark tissue during dormancy induction [6, 15]. The unknown1 sequence showed similarity to shoot and fruit peach ESTs, but this is the first report of the regulation of this gene.
During the late response, there is a large up-regulation of the defence-related genes LEA, metallothionein and PR-1. LEA proteins have the presumed role of cellular stabilizers under stress conditions. An Arabidopsis LEA domain-containing gene (At4g21020) similar to the peach gene reported here was found expressed in seeds of Arabidopsis . The increase in LEA expression can be related to the cold acclimation induced by photoperiod, as a protective measure against dehydration. This adaptation to dehydration was also previously found starting in the first weeks of SD-dormancy induced in poplar [5, 7]. In contrast, a previous study found that SD induced a down-regulation of a different LEA protein in peach bark . LEA genes have been found down-regulated during the dormancy release in raspberry  and oak buds . If the LEA gene we have identified is indeed involved in dehydration resistance or cold hardiness, the lagging LEA expression we observed in the evg mutant is consistent with the impaired cold hardiness response previously observed in seasonal LEA expression in evg and deciduous genotypes of peach .
Putative metallothioneins were found up-regulated during dormancy release in raspberry  and Norway spruce , whereas other metallothioneins were found up-regulated during dormancy development in poplar buds , in dormant cambial tissue in aspen  and during chilling accumulation in grape . Similar metallothioneins to the peach sequence found in our experiment were also expressed during fruit development in apricot and in response to cold stress in apple fruit . Several roles have been defined for metallothioneins: detoxification of heavy metals, homeostasis of essential metal ions, and regulation of gene expression in development processes.
The class 1 pathogenesis-related proteins are not only involved in plant defence responses, but also in development . However, little is known about the molecular function of class 1 pathogenesis-related proteins in plant signalling networks during development. A dual function for some pathogenesis-related proteins as antifreeze proteins during dormancy has been proposed . An increase in PR-1 expression was similarly found during dormancy entrance in poplar .
The non-dormant phenotype of the peach evg corresponds to a deletion in the LG1 group of the general genetic map [21, 23]. A cluster of DAM genes that belong to the SVP-subfamily of MADS-box genes are located in this deleted region . Three of these genes, PpDAM1, PpDAM2 and PpDAM4 are the most likely candidates for the regulation of growth cessation and terminal bud formation . In this work, two of the DAM genes, the PpDAM1 and PpDAM6, were detected and differentially expressed between WT and evg. Their expression was up-regulated after the change in photoperiod and increased continually during bud development. A SVP-like MADS-box factor similar to the PpDAM6 gene showed endodormancy-associated expression in lateral buds of Japanese apricot . Additionally, two putative SVP-like genes, with sequences similar to the PpDAM6 and PpDAM1 genes, were down-regulated during the dormancy release in Rubus idaeus L. buds . The PpDAM6 gene is induced by short photoperiods  and unpublished data from our lab shows it to be cold-suppressed. There are six peach DAM genes expressed in WT trees and all six are not expressed in the mutant evg . Here only two of the six genes we know should be definitely differentially expressed between the WT and mutant were detected with the SSH PCR technique we used in this study. This is in line with the known limited sensitivity of SSH for isolating genes like transcription factors that are expressed at low absolute levels.
The most strongly up-regulated gene after several weeks of SD photoperiod inducing-conditions was similar to the epicotyl-specific tissue protein from Striga asiatica. A similar protein in Cicer aeretinum, CanST-2, seems to have an opposite expression pattern, since its transcript level decrease when the growth of epicotyls is inhibited . However, the molecular function of the epicotyl-specific tissue protein in the bud development process remains unknown. A similar protein was found to be down-regulated by low temperatures in peach bark .
Three additional genes of unknown function were found up-regulated after several weeks of SD photoperiod. Unknown2 expression was induced by SD photoperiod and cold in other study of SD responses in peach . The unknown4 sequence showed similarity to a hypothetical protein of Vitis vinifera; however, a putative function and relationship with growth cessation or dormancy could not be assigned. The unknown3 sequence represented a novel transcript in plants. These unknown genes can now be associated with SD responsiveness in peach and may represent novel components of growth cessation and/or dormancy development in peach or other perennial species. Release of the assembled peach genome sequence (ongoing, Dr. Doreen Main, personal communication) will allow the localization of these genes in the genome and determining if they co-localize with genetic and physical map locations known to regulate phenological events such as bud set, chilling requirement, or bud break .