Apple production depends on the fulfilment of a chilling requirement for bud dormancy release. Insufficient winter chilling results in irregular and suboptimal bud break in the spring, with negative impacts on apple yield. Trees from apple cultivars with contrasting chilling requirements for bud break were used to investigate the expression of the entire set of apple genes in response to chilling accumulation in the field and controlled conditions. Total RNA was analysed on the AryANE v.1.0 oligonucleotide microarray chip representing 57,000 apple genes. The data were tested for functional enrichment, and differential expression was confirmed by real-time PCR. The largest number of differentially expressed genes was found in samples treated with cold temperatures. Cold exposure mostly repressed expression of transcripts related to photosynthesis, and long-term cold exposure repressed flavonoid biosynthesis genes. Among the differentially expressed selected candidates, we identified genes whose annotations were related to the circadian clock, hormonal signalling, regulation of growth, and flower development. Two genes, annotated as FLOWERING LOCUS C-like and MADS AFFECTING FLOWERING, showed strong differential expression in several comparisons. One of these two genes was upregulated in most comparisons involving dormancy release, and this gene's chromosomal position co-localized with the confidence interval of a major quantitative trait locus for the timing of bud break. These results indicate that photosynthesis and auxin transport are major regulatory nodes of apple dormancy and unveil strong candidates for the control of bud dormancy.

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The molecular control of bud dormancy establishment and release is still not well understood, although some genes have already been demonstrated to play important roles in this process. The dormancy-associated MADS-box (DAM) genes were first identified in the peach EVERGROWING locus and are considered the main regulators of bud dormancy control. In this work, the apple (Malus × domestica Borkh.), a perennial plant adapted to temperate climates that displays cycles of growth and bud dormancy, was screened for the presence of DAM genes. The candidate genes retrieved were characterized in comparison to DAM genes from other species. Four of them (MdDAM1-4) are structurally very similar to the reported DAM genes. When apple genomic segments containing these candidates were compared to the peach EVERGROWING locus, a highly conserved noncoding region was detected inside their largest intron. Similar sequences were also identified inside introns of apricot and pear DAM genes. Organ expression patterns revealed that MdDAM1-4 are mainly expressed in dormant buds and seeds, with low transcript accumulation in vegetative structures. In addition, the MdDAM genes showed seasonally oscillating patterns of steady-state messenger RNA (mRNA) levels and were downregulated by artificial chilling. Motif analyses in the promoter and in the intronic conserved region of the MdDAM genes disclosed some clues to the regulation of the expression patterns observed. Possible roles for the conserved intronic sequence in dormancy regulation are discussed.

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