Dehydrins are believed to play a fundamental role in the response of plants to various abiotic and biotic stresses. They make up a multigene family with 10 members in Arabidopsis [18, 35], 8 members in rice , 13 members in barley , and 11 members in poplar . However, only 4 DHN genes were identified in the published V. vinifera genome sequence [30, 47], including two YnSKn-type DHNs (DHN1 and DHN4) and two SKn-type DHNs (DHN2 and DHN3). Neither Kn- nor KS-type DHNs were found in this species, which differs from the DHN gene families from other plant species characterized to date and suggests that these types of genes may have been lost in grape species (Figure 2).
Expansion of the DHN family has generally occurred through tandem duplication events and whole-genome duplications. For example, At1g20440/At1g20450, At3g50970/At3g50980, and At4g38140/at4g39130 arose from tandem duplications, while At1g20450/At1g76180, At2g21490/At4g39130, and At3g50970/At5g66400 arose from a whole-genome duplication event in Arabidopsis , which together resulted in an increase of 6 DHN genes. Similarly, whole-genome and tandem duplication events were responsible for an increase of 3 and 2 DHN genes, respectively, in poplar . At least 3 DHN genes arose from tandem duplication events in rice , and it is possible that the two clusters of DHN genes on chromosomes 5 H and 6 H in H. vulgare, respectively , which show a high level of sequence identity within each cluster, may have resulted from tandem duplication events.
While the genomes of poplar, rice and Arabidopsis have undergone at least one recent whole-genome duplication event, the grapevine genome has not . Instead, the four grapevine DHNs have likely arisen from an ancestral genome , which is consistent with the low level of sequence similarity between the four sequences. However, DHN3 and DHN4 lay in close proximity on chromosome 3 in V. vinifera, which implies that one of them may have arisen through a tandem duplication event despite their low level of identity. Therefore, it seems that the relatively low number of DHN genes in grapevine may simply be due to a lack of duplication events in this genus. Indeed, it has been suggested that gene family expansion in grapevine has been selective, occurring mainly in those genes involved in aromatic features .
In silico characterization of V. vinifera and V. yeshanensis DHN protein sequences suggested they were all highly hydrophilic and disordered, but with distinct differences in pI, kinase specificity and content of functional motifs. The two YnSKn-type DHNs (DHN1 and DHN4) possessed a higher pI than the SKn-type DHNs (DHN2 and DHN3). Since positively charged DHN proteins bind negatively charged membranes with a high affinity , it follows that the YnSKn-type DHNs, and especially DHN1, could very well bind with the cell membrane in grapevine. It has been suggested that the binding of DHNs to membranes may be modulated by phosphorylation through an alteration of net charge . The DHN1 and DHN4 proteins from grapevine were found to contain a higher number of putative PKC sites than CK2 sites, whereas DHN2 and DHN3 bore a higher number of putative CK2 sites than PKC sites (Table 1; Figure 2). This finding is in agreement with a previous suggestion that YnSKn-type DHNs are mainly phosphorylated by PKC, while SKn-type DHNs are mainly phosphorylated by CK2 .
DHN proteins with similar physicochemical properties often also exhibit similar expression patterns. For example, while genes encoding alkaline YnSKn-type DHNs, such as At5g66400
HvDhn9 and HvDhn10, are generally induced by both embryogenesis and various types of stress [18, 23], those encoding acidic SKn- and KnS-type DHNs, such as At1g20440
HvDhn8 and HvDhn13, are expressed constitutively in vegetative tissues and are also up-regulated by various types of stress [18, 23, 35]. The expression patterns of grapevine DHN1 and DHN2 agree with those predicted by their classification, which suggests that this holds true in the species analyzed here.
Even though the grapevine DHN1 and DHN4 (YnSKn-type), as well as DHN2 and DHN3 (SKn-type), proteins are grouped into only two classes, all four members of the grapevine DHN family exhibited very distinct patterns of expression (Figure 4, Figure 5, Figure 6, and Figure 7). We found grapevine DHN1 to be induced by drought, cold, heat, E. necator, and to be expressed during late stages of embryogenesis, which corresponds well with previous reports [28, 29]. Conversely, DHN2 was found to be constitutively expressed in vegetative tissues and was up-regulated under cold and heat conditions, as well as during late embryogenesis (Figure 4, Figure 5, Figure 6, and Figure 7). In contrast, very low levels of DHN3 expression were only detected during seed development with no induction observed in vegetative tissues following any of the stress or signaling molecule treatments studied here. Correspondingly, although no DHN3 transcripts could be identified in GenBank’s EST database, a large number (tens to hundreds) of the remaining grapevine DHN genes were (data not shown), which suggests that DHN3 is expressed at undetectable levels in most tissue types. Likewise, DHN4 was also specifically expressed during late embryogenesis, but at far higher levels than DHN3 (Figure 4 and Figure 5). These results suggest that the function of the grapevine DHN genes is likely divergent, but may also exhibit some level of overlap.
The accumulation of DHNs in plants is believed to have been associated with the acquisition of desiccation tolerance in these organisms  and expression levels of these genes in vegetative tissues has generally been found to be higher in drought-tolerant cultivars than in their susceptible counterparts [48–51]. However, this is not always the case, as differences in expression levels between tolerant and sensitive genotypes are often dependent on the type of DHN, as well as the duration of the stress. While both V. yeshanensis and V. vinifera have been found to exhibit some tolerance to drought, the former exhibits a higher tolerance than the latter and also displays resistance to cold [25, 26]. In the case of induction via temperature stress, both DHN1 and DHN2 exhibited cold and heat responsiveness; however, DHN1 appeared to be far more responsive than DHN2 (Figure 6 C-F). Interestingly, induction tended to be higher in V. vinifera than V. yeshanensis, which is contrary to the levels of temperature sensitivity in these two species.
Conversely, among the four grapevine DHN genes tested, only DHN1 was induced by drought stress in vegetative tissues. This gene was up-regulated between 1–2 d after the initiation of drought conditions in V. yeshanensis, while its expression level at this time remained unchanged in V. vinifera, suggesting that the expression of DHN1 was quicker to respond to drought in the tolerant genotype. However, V. yeshanensis did not show a higher level of DHN1 expression than V. vinifera at 3 and 4 d following treatment (Figure 6 A and B). A similar situation has been observed in barley, where the HvDhn6 gene was expressed earlier in tolerant cultivars than susceptible cultivars under drought conditions, but at lower levels than the susceptible cultivars at time points that were further from the commencement of drought conditions [49, 50].
Generally, DHN genes are up-regulated under drought stress and down-regulated following rehydration [52–55]. However, in this study, the grapevine DHN1 and DHN2 genes also displayed induction 2 h post-rehydration (Figure 6 A and B). In line with this, it has been found previously that leaf ABA content increases during early phases following re-hydration . Therefore, the up-regulation of grapevine DHNs after rehydration may correspond to a change in leaf ABA content, since both genes were found to be responsive to this plant hormone (Figure 7 A and B).
Recent studies have indicated that DHNs are also responsive to pathogen infection. For example, a DHN gene can be utilized to predict blast resistance in rice , and the Arabidopsis LTI30 and RAB18 genes have been found to be up-regulated by inoculation with powdery mildew . This pathogen-induced expression of DHNs may provide another important function for this type of gene in disease resistance. In the current study, only DHN1 was found to be up-regulated in V. yeshanensis and V. vinifera following inoculation with E. necator, which is the causative agent of grapevine powdery mildew (Figure 6G). Intriguingly, the expression level of DHN1 was higher in the resistant V. yeshanensis than in the susceptible V. vinifera, and a second induction event was also apparent in V. yeshanensis that was lacking in V. vinifera. These results suggest that DHN1 may participate in powdery mildew resistance in V. yeshanensis.
DHN1 from V. vinifera was also induced by the signaling molecules SA and MeJA, which are known to be involved in defense response, providing further evidence that it could play a role in systemic acquired resistance . It has been demonstrated previously that a number of pathogen-responsive genes were up-regulated in transgenic Arabidopsis plants overexpressing DHN-5, which implies that DHNs might act as stress signaling molecules that regulate defense genes . This may also be the case for the DHN1 genes from grapevine (Figure 7A), although further experiments will be necessary to show this definitively.
The expression of stress-responsive genes depends upon the presence of cis-regulatory elements in their promoter regions , as has been shown to be the case for barley DHN genes . The four grapevine DHN promoters exhibited distinct differences in the composition and distribution of putative regulatory elements held within them. ABREs, which are one of the most common cis-elements in the DHN promoters, likely played a role in the induction of DHN1 by ABA, mediating its expression under drought conditions. Indeed, when taken together, all of the putative regulatory elements identified within both the DHN1 and DHN2 promoters could account for their up-regulation by a variety of stresses and their corresponding signal molecules (Figure 8). In contrast, relatively few regulatory elements were found in the DHN3 and DHN4 promoters, which corresponds with the fact that neither of these genes were found to be induced by any of the stresses or signaling molecules analyzed.
The quantity and location of regulatory elements could also have a profound effect on grapevine DHN expression. It has been found previously that a single copy of an ABRE is not sufficient for ABA-responsive induction of transcription . In this study, a higher number of ABRE elements were located in DHN1 and DHN2 promoters than in DHN3 and DHN4 promoters (Figure 8); correspondingly, the two former genes were responsive to induction by ABA, whereas DHN3 and DHN4 were not. Furthermore, the Skn-1 motif, which has been shown previously to confer a promoter with endosperm-specific expression , was found in all four grapevine DHN promoters. However, these motifs were located much nearer to the translational start codon in DHN1 and DHN4 promoters than in DHN2 and DHN3 promoters, which may provide an explanation for increased up-regulation of DHN1 and DHN4 during late embryogenesis (Figure 4 and Figure 5).