In plants, members of the Hsf family have been described as key regulators in molecular and cellular responses to stress conditions
[1, 7]. Furthermore, data from tomato and Arabidopsis have shown that the Hsfs are important components involved in developmental signalling
[13, 14]. Both size and composition of the Hsf family have been analyzed and characterized in different plant species
. The present study investigates for the first time this gene family in the economically relevant domesticated apple and shows that its genome contains 25 full length Hsf genes. This number is similar to that of Populus trichocarpa for which 28 loci encoding Hsf proteins were found
. Velasco et al.  have shown that genome wide duplications had occurred in apple causing the expansion of several gene classes. Indeed, it was found that the enlargement of the MdHsf family is in particular originated from segmental duplications between different chromosomes. This situation is similar in maize and in Populus, in which segmental Hsf gene duplications were more prevalent than those of tandem duplications
[9, 10]. Gene duplications have an important role not only in the genomic rearrangement and expansion but also in diversification of gene function. In particular, genes encoding for nucleic acid binding proteins, among which transcription factors, originated mostly by segmental duplication. In contrast, membrane proteins and proteins involved in the stress response are encoded by genes mainly duplicated in tandem
[18, 19]. Therefore, the prevalence of segmental duplication events in MdHsf expansion may be associated to the fact that these genes act as transcriptional regulators.
Malus, Arabidopsis and Populus belong to the Rosid lineage and they are grouped in two distinct clades, namely Fabids (Malus and Populus) and Malvids (Arabidopsis)
. It was observed in the present study that the majority of the MdHsfs had a closer phylogenetic relationship to the PtHsfs than to the AtHsfs. This may be attributable to the fact that Malus and Populus belong to the same Fabids clade, and as they are both trees may have adapted to prolonged and repeated environmental constraints, unlike Arabidopsis.
Functional diversification of multifamily duplicated genes has been observed in trees. For example, the family of the glutathione S-transferase in Populus has a clear divergence in expression patterns in response to different stress treatments
. Therefore the presence of many duplicated Hsf genes in the apple genome may be related to the fact that a sub-functionalization has taken place especially to cope with prolonged and specific stress conditions.
MdHsf genes were found to be expressed in several apple tissues. In particular, members belonging to the A1 and B1 subclasses, such as MdHsfA1a, MdHsfA1d, MdHsfB1a, MdHsfB1b, were constitutively expressed in different tissues. A similar situation was found in other plants like Arabidopsis where A1-type Hsfs were involved in house-keeping processes under normal conditions, being ready for the fast activation of other Hsfs genes following stress treatment
[22, 23]. Furthermore, expression data from flower and fruit tissues indicated that some duplicated gene pairs, e.g. MdHsfA9a and MdHsfA9b, exhibited differences in their expression levels. This suggests that they may be subjected to a different regulation in apple tissue
In contrast, the expression of MdHsfA2a and MdHsfA2b was mainly detected in full bloom flowers. AtHsfA9 and LeHsfA2a (Le, Lycopersicon esculentum) were found expressed in seed and developing pollen grains
[13, 14, 24]. It was shown that the presence of these Hsfs during plant development is important for heat shock protein activation. This suggests that MdHsfA2a and MdHsfA2b may be important during pollination and fertilization, which occurs at anthesis.
Effects of heat stress (HS) on Hsf gene expression has been examined in several plant species, but no data are available about Hsf expression in trees exposed to naturally increased temperatures. Under laboratory settings, it was shown that AtHsfA1a and AtHsfA1b regulate the early response to HS in Arabidopsis
[22, 25]. AtHsfA2 is rapidly induced by HS, and it is involved in enhancing and maintaining of HS-response when plants are exposed to prolonged or repeated cycles of HS
[26, 27]. Similarly to AtHsfA2, AtHsfA3 is involved in thermo-tolerance mechanisms
[7, 28, 29]. The A1-type MdHsfs were expressed at the same level also in leaves from plants growing in field and exposed to different temperature conditions. MdHsfA2a
c were instead strongly induced. This may suggest that these types of MdHsfs could be involved in maintaining the stress response when apple trees are exposed to prolonged periods of high temperature conditions.
In contrast to class A Hsfs, genes assigned to the B and C classes have so far not been fully characterized. Members of the B class were shown to act mainly as repressors of the expression of HS inducible genes
[30, 31]. Some of them form a complex with Hsf A-types to maintain housekeeping gene expression during HS regimes
. Therefore, the strong transcriptional activation in apple may indicate that some of them may have a role in the response to the high temperatures also in this species. For the majority of MdHsfs, increased messenger RNA levels were observed under naturally increased temperatures. However, MdHsfA9b and MdHsfB4a-b were the only Hsf genes showing low transcript abundance. Although proteomic data are not available for all MdHsfs genes, their activation or repression may suggest that these transcripts could have a high hierarchy of molecular events induced by the high temperatures.