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Dehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and also participate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHN gene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderately resistant to powdery mildew.
Four DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequence identity between the two species but little homology between the genes themselves. These genes were designated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predicted to be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number of functional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1 was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat, embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). It was expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a second round of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V. vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited no responsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetative tissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development at extremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4 exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particular cis-elements within the promoter regions of each gene was positively correlated with their expression profiles.
The grapevine DHN family comprises four divergent members. While it is likely that their functions overlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our results suggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elements in the promoter regions of the DHN genes.
Dehydrins (DHNs) are a class of hydrophilic, thermostable stress proteins with a high number of charged amino acids that belong to the Group II Late Embryogenesis Abundant (LEA) family. Genes that encode these proteins are expressed during late embryogenesis, as well as in vegetative tissues subjected to drought, low temperature and high salt conditions [1-3]. Intriguingly, over-expression of DHN genes in transgenic plants has been found to enhance resistance of the transgenic lines to various adverse environments, such as cold, drought, salinity and osmotic stress [4-7], which has raised significant interest in their putative application for crop improvement. Furthermore, it has recently been shown that reduced levels of dehydrins in transgenic Arabidopsis seeds leads to reduced seed longevity [8], emphasizing their importance to seed survival in addition to their influence on vegetative stress tolerance.
While it is generally accepted that DHNs function to protect cells from damage caused by stress-induced dehydration [9], their precise mechanism remains elusive. However, it has been proposed that they may carry out their function through membrane stabilization by acting as chaperones to prevent the aggregation and/or inactivation of proteins under dehydration or high temperature conditions [5,10,11].
Classification of DHNs is based upon structural features of the proteins, such as the presence and copy number of certain conserved motifs, such as the K-, S-, and Y-segments. To date, these proteins have been divided into 5 subclasses, including YnSKn, YnKn, SKn, Kn and KnS [12]. All DHNs possess at least one K-segment (EKKGIMDKIKEKLPG), which is generally located at the C-terminal end of the protein and has the ability to form an amphipathic helix-like structure that may play a role in its interaction with membranes and proteins [13,14]. The S-segment consists of a track of serines that can be modified through phosphorylation and may function in the regulation of protein conformation and ion-binding activity [15-17]. The Y-segment (DEYGNP) is located near the N-terminus and shows partial amino acid identity to the nucleotide binding site motif of chaperone proteins from various organisms [12].
Several other conserved regions have also been identified in a subset of DHNs. For example, lysine-rich segments (Lys-segments) contain a cluster of lysines that are generally located between the S- and K-segments [18,19] and have been suggested to participate in the binding of DHNs to DNA or RNA [19]. Nuclear localization signals (NLSs), which bear an RRKK motif, have been found specifically in YSKn-type DHNs and play a role in their localization to the nucleus [15,16,20]. Furthermore, phosphorylation has been found to be an important factor for substrate binding of DHNs [16,17,21], and recently a His switch has been found to be involved in the regulation of membrane binding of the Arabidopsis thaliana DHN, LTI30 [22].
At the functional level, DHN family members often exhibit sub-functionalization, with different genes displaying differential expression profiling throughout development and under stress conditions. For example, while both LTI29 (SK2) and LTI30 (K6) were up-regulated in Arabidopsis under low temperature conditions, only LTI30 was up-regulated following salt treatments [1]. Similarly, while ten barley DHNs were found to be up-regulated by drought, only three were up-regulated by low temperatures [23], and in Oleae europaea, although expression levels of 40kDa and 42kDa DHNs increased in response to various stressors (including dehydration, high salinity, and wounding), 16kDa and 18kDa DHNs were mainly induced by salt stress [24]. These differences in expression patterns imply functional diversification within this gene family; however, at present, the relationship between subgroup classification and expression profile is unclear [9].
Grapevine is one of the most important fruit crops in the world and while the majority of grape varieties are directly cultivated from Vitis vinifera L., this species is relatively susceptible to powdery mildew (Erysiphe necator). Conversely, V. yeshanensis is a wild species of grape native to the Yanshan mountain in Hebei province, China, that is highly tolerant to both cold and drought [25,26], and is also resistant to E. necator[27]. Previously, two highly similar putative Y2SK2-type DHN genes (DHN1a and DHN1b) were identified in V. vinifera and their expression was found to be induced by multiple types of stress, such as drought, cold and high salinity [28,29]. In this study, we aimed to identify the members of DHN gene family in V. vinifera, as well as their homologous equivalents in V. yeshanensis. In doing so, we were able to investigate the functional divergence of this gene family in these two species through comparisons of their expression profiles and putative physicochemical characteristics. Furthermore, we also assessed possible relationships between specific cis-elements within DHN promoter sequences and the regulation of their expression under various conditions.
A 280-bp fragment of a DHN cDNA was cloned from drought-treated leaves of V. yeshanensis acc. Yanshan-1 using differential display reverse transcription-PCR (DDRT-PCR; Additional file 1). Subsequently, the full-length sequence was determined using 5' rapid amplification of cDNA ends (RACE) and was termed VyDHN1 [GenBank:JF900497]. The putative protein sequence of this gene was then utilized to detect DHN genes from the published V. vinifera cv. Pinot Noir clone PN40024 genome sequence [30] via BLAST analysis. Four DHN genes were identified, all of which contained a K-segment. These genes were designated VvDHN1 (corresponding in sequence to the previously identified V. vinifera DHN1a) [GenBank:XM_03631828], VvDHN2 [GenBank:XM_002285883], VvDHN3 [GenBank:CAN73166], and VvDHN4 [GenBank:XM_002283569].
The three remaining DHN genes were cloned from V. yeshanensis acc. Yanshan-1 seed-specific cDNA using primers derived from the V. vinifera sequences and were designated VyDHN2 [GenBank:JQ408442], VyDHN3 [GenBank:JQ408443], and VyDHN4 [GenBank:JQ408444]. While only 25 amplification cycles were required to clone both VyDHN2 and VyDHN4, 40cycles were required in the case of VyDHN3. Subsequently, all four V. yeshanensis genes were amplified from genomic DNA to identify intronic regions [GenBank:JF896520, JF896556, JF896557, and JF896558, respectively]. In both species, all four DHN genes consisted of two exons separated by one intron present within the S-segment.
In terms of nucleotide similarities, virtually no sequence identity was detected between the four DHN coding sequences. However, high levels of homology were noted between matching genes belonging to the two different species, with 97% (DHN4) and 99% (DHN1, DHN2 and DHN3, respectively) identity at the nucleotide level. In terms of chromosomal localization, while VvDHN1 and VvDHN2 were located on chromosomes 4 and 18, respectively, VvDHN3 and VvDHN4 were mapped to chromosome 3 in opposite orientations (Figure (Figure11).
Protein sequences were deduced from the corresponding V. vinifera and V. yeshanensis DHN cDNA sequences, and were composed of 130–206 amino acids exhibiting 97-99% identity at the amino acid level between the two species. Both K- and S-segments were found to be highly conserved between members of the V. vinifera and V. yeshanensis DHN families, whereas remaining regions displayed relatively low amino acid identity between the four genes. Furthermore, while NLS domains were identified in both DHN1 and DHN4 proteins, a Lys-rich segment was only present in DHN2 (Figure (Figure2).2). Based on the presence and number of K-, S- and Y-motifs (Figure (Figure2),2), the four DHNs from each species were classified as either Y2SK2- (DHN1), SK2- (DHN2), SK3- (DHN3), or Y3SK2-type (DHN4) proteins (Figure (Figure2;2; Table Table11).
All members of the DHN family in the two grapevine species analyzed were found to be highly hydrophilic, with GRAVY values ranging from −0.959 to −1.527 and theoretical pIs from 5.20 to 9.36 (Table (Table1).1). DHN1 and DHN4, which were YnSKn-type DHNs, possessed a higher pI than the SKn-type DHNs (DHN2 and DHN3) in both species. In terms of acidity, our analyses indicated that DHN1 was the sole basic protein, while DHN2 was the most acidic.
Many phosphorylation sites were also predicted within each of the DHN protein sequences analyzed, with DHN1 and DHN4 containing a higher number of putative protein kinase C (PKC) phosphorylation sites than casein kinase 2 (CK2) phosphorylation sites, and DHN2 and DHN3 containing a higher number of CK2 sites than PKC sites (Table (Table1;1; Figure Figure2).2). In addition, a recently identified conserved motif (LXRXXS) phosphorylated by an Snf1-related kinase (SnRK2-10) [31] was identified in both DHN1 and DHN2 proteins.
While all four grapevine DHNs were found to be rich in disordered regions and contained relatively few helix or strand motifs (see Additional file 2), the YnSKn-type proteins (DHN1 and DHN4) displayed the highest disorder index and least helix/strand-motifs. Additionally, of the few helix motifs identified, most were located within K-segments, which is consistent with the findings of a previous study of DHN protein structure [14].
To date, DHN families from both barley and Arabidopsis have been thoroughly characterized at the genomic level [18,32-35]. Therefore, to provide a further understanding of the relationships between the V. vinifera and V. yeshanensis DHNs, we conducted a phylogenetic analysis of these genes via comparison with those from barley and Arabidopsis. Overall, we found the number of DHN genes in the grapevine species (four) to be smaller than that in either barley (thirteen) or Arabidopsis (ten). Based on our phylogenetic results, the DHNs could be divided into four groups, corresponding to YnSKn-, SKn-, Kn-, and KS-type proteins (Figure (Figure3),3), where the Arabidopsis YK-type DHN (At4g39130) was included within the YnSKn group. As expected from our classification of the grapevine DHN sequences based on the presence of various conserved segments, the grapevine DHN1 and DHN4 proteins were grouped together with the YnSKn-type DHNs of Arabidopsis and barley, while the grapevine DHN2 and DHN3 proteins were grouped with the SKn-type DHNs of Arabidopsis and barley. Interestingly, both grapevine species lacked KS- and Kn-type DHNs; groups which are present in both barley and Arabidopsis.
To elucidate the physiological functions of different members of the DHN family in V. vinifera and V. yeshanensis, the expression of DHN genes was investigated at veraison in roots, stems, leaves, seeds, and fruit peels using semi-quantitative RT-PCR (Figure (Figure4).4). Both species exhibited highly similar expression profiles for their matching DHN genes. Results suggested that under normal growth conditions, DHN1 was mainly expressed in seeds, with very low levels present in the roots. DHN2 was constitutively expressed in all tissues; however, weaker levels of expression were noted in leaves and stems than in the other organs tested. DHN3 was undetectable under the current experimental conditions, suggesting that it was not expressed, or was at levels too low to be detected using this method, in these tissues. The DHN4 genes, on the other hand, were expressed specifically in seeds. These results indicate that the four DHN genes that make up the V. vinifera and V. yeshanensis DHN gene families, respectively, exhibited very distinct expression patterns in the organs tested.
Intriguingly, reactions in which DHN1, DHN2 and DHN4 were amplified often exhibited two bands: one that corresponded in size to a spliced transcript, and one that seemed to be closer in size to the band amplified from genomic DNA (ie. containing an unspliced intron). The fact that no amplification products were obtained in negative control reactions lacking reverse transcriptase confirmed that template RNA was free from contaminating genomic DNA. Therefore, it seems that these larger transcript variants resulted from the presence of unspliced DHN transcript variants (either mRNA or pre-mRNA) within the total RNA pool.
To gain a more precise understanding of grapevine DHN expression during the process of seed development, inflorescences and berries of V. vinifera were harvested from flowering to veraison and used for qRT-PCR expression analyses. DHN1 and DHN2 were found to be expressed in floral buds even 6 d before opening of the flowers (Figure (Figure5A5A and B), after which time their transcripts decreased during the middle stages of embryogenesis, and were strongly up-regulated once again during later stages of embryogenesis. Low levels of DHN3 were detected in both mid- and late-stages of seed development (Figure (Figure5C)5C) while DHN4 transcripts were only detectable during late stages of embryogenesis, with peak expression developing just prior to veraison (Figure (Figure55D).
In an attempt to determine whether DHN1, DHN2, DHN3 and DHN4 exhibited stress-responsiveness, we analyzed the expression levels of all four genes in the leaves of three V. vinifera and V. yeshanensis plants, respectively, that had been subjected to various stress conditions using real-time qRT-PCR. Neither DHN3 nor DHN4 from either species exhibited detectable levels of expression under any of the conditions tested here, including drought, cold, heat, or E. necator infection; therefore, only DHN1 and DHN2 will be discussed further in this section. In the case of drought conditions, although we found that DHN1 was induced by this treatment in both species (Figure (Figure6A),6A), we noted slight differences between the two species. While DHN1 transcripts increased in V. yeshanensis between 1–2 d after the drought treatment began and reached a maximum induction of a 237-fold increase compared to baseline expression levels 5 d after treatment, its homologue in V. vinifera was delayed in its exhibition of a response (between 2–3 d) and reached a higher maximum induction of 366-fold compared to baseline levels. Conversely, DHN2 did not appear to respond to drought treatment in either of the two species tested (Figure (Figure6B).6B). Interestingly, transcripts of both DHN1 and DHN2 also exhibited up-regulation in both grapevine species shortly after rehydration (Figure 6 A and B), with a maximum level of expression achieved after approximately 2h.
Following cold and heat treatment, both DHN1 and DHN2 were induced in V. vinifera and V. yeshanensis. While DHN2 transcripts increased gradually between 0h and 48h following initiation of cold stress in both species, DHN1 transcripts exhibited a more sudden onset of up-regulation in response to cold beginning between 6–12h after treatment (Figure 6 C and D). In the case of heat stress, both DHN1 and DHN2 transcripts increased to maximum levels 24hours following the initiation of treatment in both species, and then subsequently decreased (Figure 6 E and F).
To determine whether grapevine DHNs were responsive to biotic stress, the levels of DHN gene expression were tested in the leaves of V. vinifera and V. yeshanensis inoculated with E. necator, which is the causative agent of grapevine powdery mildew. Results suggested that only DHN1 was induced by E. necator, whereby transcripts increased gradually to a maximum at 3 d post-inoculation (dpi) and then decreased slowly after this point in both species. Interestingly, V. yeshanensis exhibited a higher peak level of expression (18-fold increase compared to expression levels immediately prior to treatment) than V. vinifera (10-fold increase), as well as an additional sharp increase in DHN1 expression 6 dpi (Figure (Figure6G).6G). In contrast, DHN2 did not appear to respond to E. necator inoculation in either grapevine species (Figure (Figure66H).
The responses of plants to abiotic and biotic stress are generally mediated by abscisic acid (ABA) and salicylic acid (SA)/jasmonic acid (JA), respectively. To determine whether the induction of grapevine DHNs under stress conditions was related to any of these signaling molecules, DHN expression was investigated in leaves from three V. vinifera plants treated with ABA, SA or MeJA, respectively. As was the case for studies involving abiotic and biotic stress treatment, no detectable levels of DHN3 or DHN4 expression could be detected in leaves treated with any of these chemicals. While DHN1 was induced by ABA, SA and MeJA (Figure (Figure7A),7A), the most considerable up-regulation (160-fold compared to baseline levels) was noted 8h following application of ABA. DHN1 transcripts from leaves treated with MeJA reached a maximum induction of ~20-fold compared to baseline levels approximately 4h following application, whereas a similar level of induction was reached 8h following treatment with SA. In the case of DHN2 transcripts, expression reached a maximum 5-fold induction compared to baseline levels 4h after application of ABA. Although slight modifications were also noted in DHN2 expression levels following SA and MeJA applications, respectively, they differed only slightly from changes observed in untreated samples (Figure (Figure77B).
All four DHN promoters (including 1500-bp of sequence upstream of the translational start codon) were cloned from V. yeshanensis acc. Yanshan-1 [GenBank: JF899925 for VyDHN1, GenBank: JX110839 for VyDHN2, GenBank: JX110840 for VyDHN3, and GenBank: JX110841 for VyDHN4] using primers derived from the corresponding V. vinifera sequences. High levels of homology were detected between matching promoters from the two different species, with 94% (DHN1), 96% (DHN2), and 92% (DHN4) identity at the nucleotide level. The promoter of DHN3, on the other hand, only exhibited 84% identity between the two species due to a 205-bp deletion in this sequence from V. yeshanensis.
To elucidate whether the differential expression patterns of the four grapevine DHN genes correlate with transcriptional regulation via their promoters, upstream regions of each gene from V. yeshanensis and V. vinifera were scanned for putative cis-regulatory elements using the PlantCARE database [36]. Nucleotide sequences including 1500-bp upstream of each start codon were obtained from the V. vinifera cv. Pinot Noir clone PN40024 genome database [30] , while those from V. yeshanensis were obtained directly via cloning, and cis-regulatory elements were classified into three groups according to their potential responsive functions: abiotic stress-related elements, biotic stress-related elements, and seed development-related elements. Abiotic stress-related elements comprised ABA-responsive elements (ABRE), dehydration-responsive elements (DRE), heat shock-responsive elements (HSE), and low temperature-responsive elements (LTR). Biotic stress-related elements included MeJA-responsive elements (MeJA-RE), salicylic acid-responsive elements (TCA), as well as stress- and defense-responsive elements (TC-rich repeats). Seed development-related elements comprised only endosperm-specific expression elements (Skn-1 motif).
All cis-regulatory elements found in the V. yeshanensis and the V. vinifera DHN promoters, which exhibited a similar composition and distribution of putative regulatory elements between corresponding promoters, are shown in Table Table2.2. There were obvious differences in the abundance and distribution of cis-regulatory elements in the four DHN promoters analyzed (Figure (Figure8).8). The VyDHN1 and VvDHN1 promoters had the most diverse collection of putative cis-regulatory elements, including several involved in stress-response, seed development, and hormone signaling (Table (Table2).2). In the case of the VyDHN2 and VvDHN2 promoters, HSE, LTR and TCA elements were lacking when compared to VyDHN1 and VvDHN1. Only a small number of potential cis-elements were identified in the VyDHN3, VvDHN3, VyDHN4 and VvDHN4 promoters. In the VvDHN3 promoter, an ABRE, Skn-1 and two TC-rich elements were scattered evenly throughout the promoter, while the ABRE element was absent in that of VyDHN3. In VvDHN4, two ABREs, a Skn-1 and a MeJA-RE were concentrated in the 3' region of the promoter, while a TCA element was present in the 5' region. A similar distribution was noted in the VyDHN4 promoter, with an additional Skn-1 motif present 765-bp upstream of the translational start codon.
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 [45], 13 members in barley [23], and 11 members in poplar [46]. 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 (Figure22).
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 [18], 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 [46]. At least 3 DHN genes arose from tandem duplication events in rice [45], and it is possible that the two clusters of DHN genes on chromosomes 5H and 6H in H. vulgare, respectively [32], 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 [30]. Instead, the four grapevine DHNs have likely arisen from an ancestral genome [30], 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 [30].
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 [13], 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 [22]. 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 (Table1;1; Figure Figure2).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 [22].
DHN proteins with similar physicochemical properties often also exhibit similar expression patterns. For example, while genes encoding alkaline YnSKn-type DHNs, such as At5g66400HvDhn1HvDhn2HvDhn3HvDhn4HvDhn7HvDhn9 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 At1g20440At1g20450At1g76180At1g5410HvDhn8 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 (Figure4,4, Figure Figure5,5, Figure Figure6,6, and Figure Figure7).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 (Figure4,4, Figure Figure5,5, Figure Figure6,6, and Figure Figure7).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 (Figure44 and Figure Figure5).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 [12] 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 (Figure66 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 2h 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 [56]. 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 [57], and the Arabidopsis LTI30 and RAB18 genes have been found to be up-regulated by inoculation with powdery mildew [18]. 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 (Figure6G).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 [58]. 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 [11]. This may also be the case for the DHN1 genes from grapevine (Figure (Figure7A),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 [59], as has been shown to be the case for barley DHN genes [32]. 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 (Figure8).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 [59]. In this study, a higher number of ABRE elements were located in DHN1 and DHN2 promoters than in DHN3 and DHN4 promoters (Figure (Figure8);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 [60], 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 (Figure44 and Figure Figure55).
The DHN gene family was identified in a genome-wide search of the published genome sequence of V. vinifera, and the corresponding homologues were isolated from V. yeshanensis. A large expansion of the DHN family apparently did not take place in grapevine, although it has been a common occurrence in other plants. The four grapevine DHN genes shared a low sequence identity, and exhibited clear differences in physicochemical properties and expression profiles, which indicates functional diversification within the grapevine DHN family. DHN1 appeared to be the principal stress-responsive gene in grapevine species, and was induced not only by various abiotic stresses but also by E. necator. The small size and distinct expression profiles of the grapevine DHN gene family makes it an excellent model to elucidate functional differentiation within this gene family, which should contribute to the further understanding of these genes in plants.
V. yeshanensis acc. Yanshan-1 and V. vinifera cv. Pinot Noir were obtained from the Grapevine Repository of Northwest A&F University, Yangling, Shaanxi, China. One-year old rooted seedlings of Yanshan-1 and Pinot Noir were maintained in a greenhouse, and were utilized for stress experiments. For expression analysis in different plant tissues, root, stem, leaf, seed and fruit peel samples were harvested from three representative veraison-stage V. vinifera and V. yeshanensis plants, respectively, that had been grown in the field. For expression analysis during seed development, flower buds were harvested from three Pinot Noir plants grown in the field 6days before flower opening (−6 daf). Flowers were collected at flower opening (0 daf) and young berries were harvested at 6 daf. In addition, seeds were collected from 12–66 daf. Samples were frozen in liquid nitrogen and stored at −80°C.
Total RNA was isolated from plant tissues using the method described by Reid et al. [61]. Subsequently, RNA was treated with 10 units RQ1 RNase-free DNase (Promega, Shanghai, China) in the presence of 100 units RNase inhibitor at 37°C for 30min, followed by extraction with phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol (24:1). RNA was then precipitated with ethanol and dissolved in RNase-free water. First-strand cDNA synthesis was carried out using 1μg total RNA and the PrimeScript™ RT reagent Kit (TaKaRa Biotechnology, Dalian, China) with 2.5μM oligo dT primer and 2.5μM random 6mer primer. The reaction mixture was incubated at 37°C for 40min and the reverse transcriptase was then inactivated at 85°C for 5s.
A fragment of the VyDHN1 coding sequence was amplified from drought-treated leaves of V. yeshanensis acc. Yanshan-1 using differential display reverse-transcription PCR (DDRT-PCR) as described by Lin et al. [62]. Briefly, first-strand cDNA was synthesized from 1μg total RNA isolated from leaves subjected to drought at 0, 2, 3, 4, and 5 d following onset of treatment using primer T11VA (V=A, C, G) at 37°C for 1h with 200 units of M-MLV (Promega) according to the manufacturer's instructions. This was followed by PCR amplification with primers T11VA and S476 (CCAAGCTGCC), followed by separation on a 6% polyacrylamide gel. Differential fragments were recovered, amplified by a second round of PCR using the same parameters as the first, and cloned into the pGEM-T easy vector (Promega). The 5' end of the VyDHN1 cDNA was obtained by 5' rapid amplification of cDNA ends (5' RACE) using the BD SMART RACE cDNA Amplification Kit (Clontech, CA, USA) with primer VD1-GSP1 (see Additional file 3 for primer sequence) according to the manufacturer's recommendations. The full-length VyDHN1 sequence was then deduced through alignment of the original DDRT-PCR fragment and 5' RACE sequence.
The encoded protein sequence of the VyDHN1 gene was used to identify four V. vinifera DHN genes containing putative K-segments via BLAST analyses. BLAST analyses were also performed against the predicted grapevine DHN genes using ten previously identified Arabidopsis DHN proteins as query sequences [18]. These results were further validated by searching for Vitis DHN sequences in the Pfam database [63].
For the remaining DHN genes, seeds were harvested from V. yeshanensis at veraison. The DHN genes were amplified from cDNA using PrimeSTAR® HS DNA polymerase (TaKaRa) with primers that were designed based upon the DHN genes of V. vinifera cv. Pinot Noir (see Additional file 3). Cycling parameters for amplification of VyDHN2 and VyDHN4 were as follows: 94°C for 3min, 25cycles at 94°C for 30s, 60°C for 30s and 72°C for 30s, followed by a final elongation at 72°C for 5min. The same parameters were utilized in the case of VyDHN3 except that 40cycles were necessary for its amplification. Subsequently, all four V. yeshanensis DHN genes were also amplified from genomic DNA to identify intronic regions using the same primers.
V. yeshanensis DHN promoters, including 1500-bp of upstream sequence in each case, were amplified from genomic DNA using PrimeSTAR® HS DNA polymerase (TaKaRa) with primers that were designed based upon the DHN genes of V. vinifera cv. Pinot Noir (see Additional file 4). Cycling parameters were as follows: 94°C for 3min, 30cycles at 94°C for 30s, 60°C for 30s and 72°C for 2-3min, followed by a final elongation at 72°C for 5min. PCR products were cloned into pGEM-T easy (Promoga) and three clones were sequenced per DHN gene.
Drought experiments were conducted by withholding water from V. yeshanensis and V. vinifera seedlings. Leaves were harvested 0, 0.5, 1, 2, 3, 4, 5, and 6 d following onset of treatment. Subsequently, the stressed plants were watered to soil saturation and leaves were collected 0.25, 0.5, 2, 6, 12, 24, and 48h after watering. Plants grown under a normal watering regime were used as a control. For cold- and heat-stress induction, V. yeshanensis and V. vinifera seedlings were maintained in a growth chamber at either 4°C or 40°C and leaves were harvested 0, 2, 6, 12, 24 and 48h after treatment. Mock-treated control plants were kept in a growth chamber at 22°C. Pathogen treatment was carried out by inoculating the leaves of V. yeshanensis and V. vinifera with E. necator as previously described [27]. Prior to inoculation, leaves were sprayed with sterile water, and leaves were harvested 0, 0.5, 1, 2, 3, 4, 5, and 6 d after inoculation. Control plants simply underwent the sterile water spray and were not inoculated.
For signaling molecule treatment, 100μM ABA [29], 100μM SA [64], and 50μM MeJA [65] were sprayed on the leaves of V. vinifera and leaf samples were harvested 0, 1, 2, 4, 6, 8h post-treatment. Leaves sprayed with 0.05% Tween 20 solution were used as a negative control. All stress-induction experiments were performed on three independent plants for each treatment.
Semi-quantitative RT-PCR was performed using Premix Ex Taq® Version2.0 (TaKaRa) and DHN-specific primers that were designed to anneal to either side of an intron in both grapevine species (see Additional file 5 for primer sequences). Genomic DNA was utilized as a size control for unspliced transcripts. Reactions lacking reverse transcriptase were utilized as a negative control to exclude DNA contamination. Each experimental reaction (25μl final volume) contained 1μl of template cDNA along with 400 nM of each primer. Cycling parameters were as follows: 94°C for 3min, 25cycles at 94°C for 30s, 60°C for 30s and 72°C for 30s, followed by a final elongation at 72°C for 5min. The actin1 transcript [GenBank:AF369524] was utilized as an internal control using primers designed to anneal to both V. vinifera and V. yeshanensis sequences and the same general parameters as the DHN transcripts, except 20cycles were utilized for amplification. PCR products were separated on a 1.5% agarose gel containing ethidium bromide, and photographed using a Bio Imaging System (Syngene, Cambridge, UK). At least two technical replications were conducted in each case.
Real-time quantitative RT-PCR was conducted via the ΔΔCT method using the SYBR® Premix Ex Taq II Kit (TaKaRa) with primer pairs designed to anneal within the second exon of each DHN gene in both grapevine species (see Additional file 6 for primer sequences). The reactions were carried out in triplicate using 1μl template cDNA in a final volume of 25μl in an iQ5 Real Time PCR System (Bio-Rad, CA, USA) with the following thermal parameters: 95°C for 10s, followed by 40cycles of 94°C for 5s and 60°C for 30s, with a final melting gradient from 60°C to 95°C at a rate of 1°C per min. The grapevine actin1 gene was utilized as an internal control. Relative expression levels of each DHN gene from both species were analyzed using the IQ5 software and were denoted as the fold-difference from expression present at baseline levels. Paired t-tests were conducted using Origin Pro 8.0 software (OriginLab Corp., Northampton, MA, USA) to assess the significance of expression level differences between treated samples and the mock controls. Differences were deemed significant at p<0.05. Baseline expression signifies that which was present prior to treatment (ie. the first time point where relative expression is set to 1), except in the case of DHN3, where no expression was noted until 18 daf and therefore this time point was utilized as the baseline.
Chromosomal locations of VvDHN genes were predicted using the BLAT server through the Genoscope Genome Browser (http://www.genoscope.cns.fr/blat-server/cgi-bin/vitis/webBlat). To identify putative cis-acting elements within the V. vinifera DHN promoters, contigs containing the respective genes were obtained using BLAST. Intergenic regions between the DHN genes and their upstream genes were determined according to annotations provided in GenBank. In the case of V. yeshanensis DHN promoters, sequences were obtained directly by cloning. The presence of regulatory elements in 1500-bp of sequence upstream of each translational start codon was determined using the PlantCARE database [36].
Protein MW (molecular weight), pI (isoelectric point) and GRAVY (grand average of hydropathy) were predicted using the ProtParam program (Expasy tools) based on their amino acid compositions. Predictions of intrinsic disorder within each DHN gene from both species were conducted using the PONDR-FIT tool [66]. Protein secondary structures were predicted using the PSIPRED v3.0 program [67]. The sequence algorithm NetPhosK (Expasy), with its probability limit set to 60%, was utilized to predict phosphorylation sites in VvDHN and VyDHN proteins.
Phylogenetic analysis was carried out by performing multiple alignments of full-length DHN protein sequences from V. viniferaV. yeshanensisH. vulgare and Arabidopsis using MEGA5 [68]. DHN sequences from Arabidopsis and barley were obtained from previous reports [18,32-34]. An unrooted dendrogram was constructed based on the alignment with PhyML using the maximum likelihood method [69].
ABA, Abscisic acid; ABRE, ABA-responsive element; CK2, Casein kinase 2; Daf, Days after flowering; DDRT-PCR, Differential display reverse-transcription PCR; DHN, Dehydrin; Dpi, Days post inoculation; DRE, Dehydration-responsive element; HSE, Heat shock-responsive element; LTR, Low temperature-responsive element; MeJA, Methyl jasmonate; MeJA-RE, Methyl jasmonate-responsive element; NLS, Nuclear localization signal; PKC, Protein kinase C; qRT-PCR, Quantitative reverse-transcription PCR; SA, Salicylic acid; SnRK, Snf1-related kinase.
YY contributed to the design of the study, conducted the majority of experiments and drafted the manuscript; MH contributed to the powdery mildew treatment experiment; ZZ contributed to the signaling molecule treatment experiment; SL contributed to the seed development experiment; YX and CZ were involved in the design of the study and preparation of the manuscript; SDS participated in analysis of results and preparation of the manuscript; YW conceived, designed and directed the study and contributed to the preparation of the manuscript. All authors read and approved the final manuscript.
Cloning of the DHN1 coding sequence from drought-treated leaves of V. yeshanensis .
Structure prediction of DHN proteins from V. yeshanensis and V. vinifera .
Sequence of primers used for cloning DHN genes in grapevine.
Sequence of primers used for cloning DHN promoters from V. yeshanensis .
Sequence of primers used for semi-quantitative RT-PCR in grapevine.
Sequence of primers used for real-time qRT-PCR in grapevine.
This work was supported by the National Natural Science Foundation of China (No.30971972) Nonprofit Finance Fund, as well as earmarked funds for Modern Agro-industry Technology Research System (No. CARS-30-yz-7) and Research of Non-profit Service (Agriculture Section) (No. 200903044–4).
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Innovative and improved gluten-free breads and flours now taste more like their wheat-based counterparts.
For many reasons, from celiac disease to gluten intolerance to the paleo lifestyle trend and best-selling books, such as Wheat Belly and Grain Brain, people are choosing to remove gluten from their diets. "Therefore, the market for gluten-free foods is booming," said Jason May, owner of California-based Great Low Carb Bread Company. In fact, by 2020, the market is projected to be valued at $23.9 billion U.S.
The company sells a number of paleo items, which are both gluten free and low carb. "These items continue to do well for us, and we are responding to these sales trends and customer demand by continuing to develop new SKUs to add to our paleo line."
"The market for gluten-free breads and flours is experiencing robust growth and has mainstreamed more quickly than categories that start as specialty foods usually do," agreed Category Manager David Rosenberg, with Illinois-based Living Now. "With this comes a broad range of quality, with some products being nutritious, natural offerings, and others being relatively less nutritious because of certain ingredients, such as an excess of sugar or sodium. Products have become more innovative with a wider variety of offerings and improved formulations that are closer in taste to their traditional wheat-based counterparts."
Alfredo Avila president of DeLand Bakery & DeLand Natural Market in Florida, said the company has also seen significant growth in gluten-free products. "DeLand Bakery can confirm these statistics from their store sales at DeLand Natural Market: 30 percent of all daily purchases are gluten-free products."
The interest is broad, Avila added. "We are noticing more people, many Baby Boomers, interested with their health and well-being, especially with health care cost increasing. With mass media reporting the benefits of good eating habits and exercise, we are also seeing an increasing trend of young people between 18 and 30 years of age that are generally concerned with what they put into their bodies."
Market Impact
Peggy Sutton, founder and president of To Your Health Sprouted Flour Co., in Alabama, said where gluten-free flours and breads are concerned, she believes the biggest positive impacts made in recent years include taste and variety. "Many manufacturers have figured out how to make great tasting breads and baked goods with a variety of gluten-free flours. In fact, To Your Health offers 11 different sprouted gluten-free flours and 18 additional sprouted grains, legumes, seeds and other products," she said. This year, the company plans to introduce gluten-free baking mixes.
In addition, seeing more paleo items enter the market has been a positive change, said May. "To me, this is an indication that people are not only eliminating their gluten intake, but they are lowering their sugar consumption as well."
And more awareness of food sensitivities by the public has certainly impacted the market as well. "Food sensitivities can manifest in subtle ways that are difficult to diagnose, so many people have turned to elimination diets to see if that helps," Rosenberg pointed out. "Because of the wide visibility of gluten-free diets and rapid expansion of available products, consumers find it relatively easy to explore gluten-free diets to see if they feel better. From mainstream and natural food labels and supermarket shelf talkers, to restaurants offering gluten-free menu items, to articles in mainstream press and even comedians making jokes on late night television, awareness of gluten-free diets is nearly universal."
Trends in the market for gluten-free items cover the gamut of allergy friendly, lifestyle diets and more. Now many manufacturers are participating in or considering non-GMO (genetically modified organism) status for their products, noted Sutton. "Certified organic products, like To Your Health's products, already meet this criteria, but for conventional products, non-GMO certification seems to be a way to increase sales and maintain low product prices. The cost of purchasing organic ingredients and obtaining/maintaining organic certification may not be a feasible option, or even an entertained option, for some conventional manufacturers."
And since those who are sensitive to gluten can also have other food allergies, manufacturers are seeing more brands eliminate other potential allergens from their foods, Rosenberg said. "Consumers are looking for products such as Living Now that are made without soy, eggs, dairy, peanuts and other allergens to accommodate their specific dietary needs."
Along with these demands, consumers are also looking for healthier options with natural ingredients but still expect a great-tasting product. "The variety of products hitting the market continues to expand, but clean labeling is becoming a higher priority," said Rosenberg. "This means non-GMO ingredients, no artificial colors or preservatives and more organic options."
At To Your Health, the company's product line has increased largely as a result of customer requests. However, Sutton said the company also looks at very popular items in the market place, and ask whether or not sprouted versions would be favorably accepted. The company's newest sprouted gluten-free products include, rolled oats, cornmeal, corn grits, popcorn, a variety of sprouted seeds and almonds and a baking mix.
Mastering Manufacturing: Quality and Cost
It can be a challenge, formulating healthy, nutritious products that taste good and are easy to work with, but it can be done with time, expertise and commitment. "For Living Now, we looked at favorite foods like pancakes and brownies, and found ways to take out bad or questionable ingredients and make natural mixes that are made without gluten and other common allergens and are non-GMO," Rosenberg said. "Most importantly, these products had to taste great. A chef rather than scientists created our products, and we think our Living Now mixes taste even better than the conventional ones containing gluten. In fact, we get that type of positive feedback at the gluten-free shows we sample at. In addition, people who have, gluten sensitivities typically have other food sensitivities as well, so making them without common allergens in a dedicated facility better serves the consumer, but again, that takes expertise and commitment.
Avila said that considering the robust gluten-free growth, DeLand Bakery's concern is supply and demand. "With the demand of gluten-free products comes competition for the best quality raw materials," he noted. "When I mention the quality, we use non-GMO grains, so the competition will be fierce."
"DeLand Bakery is interested, regarding the gluten-free product line, in using the best ingredients we can buy when producing a delicious and nutritious product," Avila continued. "We would like to see more growers produce more certified, good-quality grains and raw material so we can continue to grow and produce our product range."
At Great Low Carb Bread Company, May said the company strives to bake high-quality products using all natural and non-GMO ingredients without adding all sorts of inexpensive and low-quality "junk fillers" that raise sugar content. "We have been perfecting our baking formulations for over a decade, and I believe this experience helps drive our success."
Sutton agreed that manufacturers have cleared the taste hurdle, but that there remains the challenge of making finished products that contain clean, more healthful ingredients to truly impact the gluten-free market in a positive way. "To Your Health provides its organic, whole grain, true-sprouted products to many manufacturers of gluten-free products who are becoming the backbone of a possible new paradigm shift to products with ingredients you can pronounce and that make a difference to the ever-growing more discerning food shopper," she said.
Along with finding the best raw materials, there is the issue of the cost of gluten-free products. "You can always fine-tune the production side of the process with new equipment and working procedures," Avila noted. "Deland Bakery strives to manufacture the highest quality product, accompanied by its certification, and to pass down healthy, nutritious products to the consumer for the best price."
Healthiest Options
Health benefits for celiac sufferers and other people with gluten intolerance are obvious. Gluten has been linked to immune system issues, leaky gut syndrome, as well as other ailments. Recent studies done by the American Journal of Gastroenterology showed that many people who don't have celiac disease are still abiding by gluten free diets for the overall health benefits.
Living a gluten-free lifestyle can be a healthy choice but it depends on the products being chosen. "It's all about balance and making the right choices," said Rosenberg. "Cookies and cupcakes are treats, and just because they're gluten free doesn't make them calorie free. For consumers and retailers, the most important thing is to look for ingredients that are natural and pronounceable and free of ingredients that may cause digestive distress."
By using high quality, non-GMO ingredients, such as almond flour, flax meal, oat fiber and all-natural stevia. "We produce high-protein, high-fiber foods that won't raise your blood sugar," said May. "Our products tout an impressive nutritional profile—we even have the lab reports to prove it."
Sprouted grain bread uses the whole grain, the process also brings out the individual subtle flavors and sweetness of each grain, legume and seed, removes bitterness, increases digestibility and increases the nutrients available. "All of To Your Health's products are certified organic and are True Sprout certified," said Sutton. "We do not add to, nor do we take away from the whole grain goodness of our gluten-free grains and flours. Our gluten-free products are manufactured in our dedicated gluten-free facility.
In-store Promoting
Retailers should make sure the offerings are clearly merchandised—visible signage that shouts out gluten free is imperative, said Rosenberg. Calling out other allergen-free qualities, such as dairy-free or egg-free is a huge plus and helps differentiate product offerings that can give your store a competitive advantage. "As the gluten-free market matures, allergen-free products will continue to become more prominent," he added." Using promotional vehicles, such as circulars, couponing, demos and in-store classes helps reach out and educate this growing market." Living Now offers a wide range of support materials to help retailers highlight gluten-free and allergy-friendly products.
Great Low Carb Bread Company products should be marketed as gluten free, sugar free and low carbohydrate. Although the company does not currently offer marketing collateral to retailers, May said they are interested in developing materials for retailers in the future.
As for To Your Health Sprouted Flour Co., the company has established an educational web site, www.truesprout.org, to inform customers about the benefits of sprouted flour. While still in its early stages of development, the website will soon also offer a catalog of recipes and baking tips, identification of true-sprouted products on the market, and where to find them.
"Being organic in addition to those benefits adds 'health-ability' to our products, too," Sutton added. To Your Health gluten-free flours are usually prominent on the baking aisles of most large retail stores and sometimes can be found in the gluten-free sections of small or conventional grocery stores. "Our rolled oats and grits can be found on many cereal aisles, and our sprouted granola, seeds and almonds most often find their home on the snack aisles of stores. We work diligently to ensure that our retailers' customers are informed, to help our retailers maximize their sales, and to ensure them that we are easy to work with and available to assist them." GFR
For More Information:
Authentic Foods, (800) 806-4737
DeLand Bakery, (386) 734-7553
Great Low Carb Bread Company, (818) 505-9499
Living Now, (888) 669-3663
To Your Health Sprouted Flour Co., (334) 584-7875

subset: commoncrawl


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Participating in a GSC Service-Learning Program or SOS Fellowship Program is a unique way to explore a new country and culture while also making a significant contribution to a local community. Ranging from two-week volunteer experiences to longer-term programs of nine weeks to 6 months and year-long international fellowships, GSC's international volunteer programs appeal to volunteers with a wide variety of ages, skills and backgrounds.
What sets us apart among choices you have in volunteering abroad?
A respected leader in international volunteerism since 1993, GSC has partnered with numerous respected development organizations to provide support to communities that need it most. Working in collaboration with international NGOs such as Heifer International and Programs for Appropriate Technologies in Health (PATH), and multi-lateral organizations including the UN Food and Agriculture Organization (FAO), GSC has developed and shared its institutional knowledge to design meaningful and effective community training programs. GSC has gained NGO status in Tanzania in recognition of its contribution to this country.
As a GSC program participant, you have the opportunity to be a part of this international development work that will benefit the community and enrich you in ways you will always remember. Your GSC volunteer contribution not only offsets your preparation and project costs, it helps support this ongoing development work and your community hosts who offer so much to you.
Read GSC program participants' stories about their experiences on their community training projects, their local in-country counterparts, and their homestay hosts.


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Brecon Estate
7450 Vineyard Drive
Paso Robles, CA 93446
11am-5pm daily.
Mailing Address
PO Box 1506
Templeton, CA 93465
Brecon Estate is located approximately halfway between San Francisco and Los Angeles, nestled in the rolling hills of the Central Coast.
North or South from Highway 101, take Highway 46 West, after 4.5 miles turn right onto the Iconic Vineyard Drive then after 5.6 miles Brecon Estate will be on your right.


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A little vintage inspiration from some of my favourite ladies today, wearing winter's must-have; fur. Pure elegance with a drop of Hollywood glamour thrown in for good measure. Delightful.
p.s. bought the most incredible burgundy suede boots this week complete with gold, glittery heels. I'm like a magpie this time of year - anything sparkly and I can't resist! xx


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6.2 We must obtain up to date evidence of your identity as soon as practicable if we have not already done so. In the event that we ask you for satisfactory proof of identification but you do not provide us with the evidence we require, we reserve the right to charge for any additional identity checks we deem necessary or, in the alternative, to terminate our instructions or business relationship.
6.3 We have a duty to report to the National Crime Agency ("NCA") if we know, or have reasonable cause to suspect, that another person is involved in money laundering. Failure on our part to make a report where we have knowledge or reasonable grounds for suspicion would constitute a criminal offence.
6.4 The offence of money laundering is defined by section 340(11) of the Proceeds of Crime Act 2002 and includes concealing, converting, using or possessing the benefits of any activity that constitutes a criminal offence in the UK. It also includes involvement in any arrangement that facilitates the acquisition, retention, use or control of such a benefit. This definition is very wide and would include such crimes as:
• deliberate tax evasion;
• deliberate failure to inform the tax authorities of known underpayments or excessive repayments;
• fraudulent claiming of benefits or grants; or
• obtaining a contract through bribery.
6.5 We are obliged by law to report instances of money laundering to NCA without your knowledge or consent. In consequence, neither the firms' principals nor staff may enter into any correspondence or discussions with you regarding such matters.
6.6 We are not required to undertake work for the sole purpose of identifying suspicions of money laundering. We shall fulfil our obligations under the Proceeds of Crime Act 2002 in accordance with the guidance published by the Consultative Committee of Accountancy Bodies.
6.7 You agree that you will co-operate with us to enable us to comply with these obligations, and that you will have no claim against us or any of our partners or staff for any loss suffered by you or any other person directly or indirectly as a result of steps taken by any of us which we believe are necessary to comply with our legal obligations.
7. Professional Rules and Practice Guidelines
7.1 We comply with the bye-laws, regulations and ethical guidelines of the Institute of Chartered Accountants in England and Wales and accept instructions to act for you on the basis that we will act in accordance with those guidelines. A copy of these guidelines can be viewed at www.icaew.com/regulations
7.2 Where you give us confidential information we shall at all times keep it confidential, except as required by law or as provided for in regulatory, ethical or professional pronouncements applicable to this engagement.
8. Conflict of Interest and independence
8.1 We have a professional obligation to act in your best interests, and so cannot act for one client in a matter where there is an actual or significant risk of a conflict with the interests of another client for who we already act.
8.2 However, we reserve the right during our engagement with you to deliver services to other clients whose interests might compete with yours or are or may be adverse to yours, subject to 8 below. We confirm that we will notify you immediately should we become aware of any conflict of interest involving us and affecting you unless we are unable to do so because of our confidentiality obligations. We have safeguards that can be implemented to protect the interests of different clients if a conflict arises. Where conflicts are identified which cannot be managed in a way that protects your interests then we regret that we will be unable to provide further services.
8.3 If a conflict of interest should arise, either between two or more of our clients, or in the provision of multiple services to a single client, we will take such steps as are necessary to deal with the conflict. In resolving the conflict, we would be guided by the code of ethics of the Institute of Chartered Accountants in England and Wales which can be viewed at www.icaew.com/regulations
8.4 In accordance with common procedures used by accountancy practices, we operate a 'Chinese wall' procedure under which our corporate finance department conducts its activities entirely separately from the department of the firm which provides services to you under this engagement. You agree that we will not be obliged to disclose to you or take into consideration in providing services to you information the disclosure of which would or might be a breach of duty or confidence to any other person.
9. Probate services
9.1 In the unlikely event that we cannot meet our liabilities to you, you may be able to seek a grant from ICAEW's Compensation Scheme. Generally, applications for a grant must be made to ICAEW within 12 months of the time you become aware, or reasonably ought to have been aware of the loss. Further information about the scheme, and the circumstances in which grants may be made, is available on ICAEW's website: www.icaew.com/probate.
9.2 If you would like to talk to us about how we can improve our service to you, or if you are unhappy with the service you are receiving, please let us know by contacting the Head of Finance and Administration John Coombs. We will consider carefully any complaint that you may make about our probate services as soon as we receive it and will do all we can to resolve the issue. We will acknowledge your complaint within five business days of its receipt and endeavour to deal with it within 8 weeks. Any compliant should be submitted to us in writing.
9.3 If we do not deal with it within this timescale or you are unhappy with our response you may of course take the matter up with our professional body, ICAEW, and the Legal Ombudsman. Complaints to the Legal Ombudsman should be made within six years of the act or omission or within three years of you becoming aware of the issue, and in either case within six months of our written response to your complaint to us. The contact details for the Legal Ombudsman are:
Letter: Legal Ombudsman, PO Box 6806, Wolverhampton, WV1 9WJ,
Telephone: 0300 5550333
10. Quality of Service and Complaints Handling Procedure
10.1 We aim to provide a high quality of service at all times. If you would like to discuss with us how our service could be improved or if you are dissatisfied with the service you are receiving please let us know by contacting the partner in charge of your affairs or John Coombs or Lynn Woodward, in our Exeter Office.
10.2 We undertake to look into any complaint carefully and promptly and to do all we can to explain the position to you. If we do not answer your complaint to your satisfaction you may take up the matter with the Institute of Chartered Accountants in England and Wales.
10.3 As part of our ongoing commitment to providing a quality service, our files are periodically subject to an independent regulatory or quality review. Our reviewers are highly experienced and professional people and are, of course, bound by the same requirements of confidentiality as our principals and staff. We reserve the right to disclose our files to regulatory bodies in the exercise of their powers.
10.4 In order for us to provide you with a high quality service on an ongoing basis it is essential that you provide us with relevant records and information when requested, reply to correspondence in a timely manner and otherwise follow the terms of the agreement between us set out in this Terms of Business and associated letter(s).
11. Terminating our Agreement
11.1 You may withdraw your instructions to us or cancel your agreement with us at any time. We ask that this is done in writing. We shall be entitled to charge you for the work we have done for you up until the date of termination.
11.2 We are entitled to keep all your papers and documents if there is any money owed to us when the matter concludes.
11.3 We reserve the right to cancel the engagement between us with immediate effect in the event of:
• your insolvency, bankruptcy or other arrangement being reached with creditors;
• failure to pay our fees by the due date(s);
• either party being in breach of their obligations where this is not corrected within 30 days of being asked to do so.
11.4 In addition, we may terminate our engagement by giving you reasonable prior written notice if we decide for whatever reason that we are no longer willing to act for you. In such circumstances you will remain liable for our charges until the date on which we cease acting for you.
12. Client Monies
12.1 We may, from time to time, hold money on your behalf. Such money will be held in trust in a client bank account, which is segregated from the firm's funds. The account will be operated, and all funds dealt with, in accordance with the Clients' Money Regulations of the Institute of Chartered Accountants in England and Wales.
12.2 In order to avoid an excessive amount of administration, interest will only be paid to you where the monies would give rise to annual interest in excess of £25 and they are held for a reasonable period of time (details of the time and amount meeting this definition are available on request). In such cases a separate client account will be opened in the name of the firm, designated as a client bank account. Any such interest would be calculated using the prevailing rate applied by NatWest Bank Plc for small deposits subject to the minimum period of notice for withdrawals. Subject to any tax legislation, such interest will be paid gross.
13. Internet / Electronic Communication
13.1 Unless you instruct us otherwise we may, where appropriate, communicate with you and with third parties via email or by other electronic means. However, internet communications are capable of data corruption and therefore we do not accept any responsibility for changes made to such communications after their despatch. It may therefore be inappropriate to rely on advice contained in an e-mail without obtaining written confirmation of it. We do not accept responsibility for any errors or problems that may arise through the use of internet communication and all risks connected with sending commercially sensitive information relating to your business are borne by you. If you do not agree to accept this risk, you should notify us in writing that e-mail is not an acceptable means of communication.
13.2 It is the responsibility of the recipient to carry out a virus check on any attachments received.
14. Fees, Charges and Payment
14.1 Our charges for the services we provide to you are computed mainly on the basis of fees for the time spent on your affairs (which depends on the levels of skill and responsibility involved) and disbursements incurred in connection with the engagement.
14.2 We will charge you reasonable disbursements incurred in the course of providing the services, which may include travel, accommodation and other expenses.
14.3 If work is required which is outside the scope of the letter, for example dealing with HM Revenue & Customs enquiries into the tax return, then this will be a separate engagement for which additional fees will be chargeable.
14.4 We will issue invoices at intervals during the course of the year.
14.5 We will add Value Added Tax, if applicable, at the rate applicable when the fee notes are raised.
14.6 We revise our rates from time to time, and details of rates in force at any time are available on request.
14.7 Our terms relating to payment of amounts invoiced (fees and disbursements) and not covered by standing orders, where appropriate, are strictly 30 days net. We reserve the right to charge interest at the rate of 1% per month (APR 12.68%) in the case of overdue accounts, accruing on a daily basis from the due date until date of actual payment. You are liable to pay the interest together with any overdue amount. This clause shall not apply to payments which you contest in good faith.
14.8 Notwithstanding clause 13.7, we may, at our absolute discretion, alternatively claim interest under the Late Payment of Commercial Debts (Interest) Act 1998.
14.9 In addition, we may terminate our engagement and cease acting if payment of any fees billed is unduly delayed. However, it is not our intention to use these arrangements in a way which is unfair or unreasonable.
14.10 We reserve the right to charge you reasonable costs and expenses incurred as a result of you not complying with our payment terms including charges for preparing and sending you reminder letters and expenses of tracing you and enforcing our terms.
14.11 In the event that this firm ceases to act in relation to your affairs you agree to meet all reasonable costs of providing information to your new advisers. In particular you agree to meet these costs even where we are required by law to provide information to a successor firm. Where a third party has agreed to pay or contribute to our charges, you nevertheless remain primarily responsible for payment of our invoices.
15. Commissions or Other Benefits
15.1 In some circumstances, commissions or other benefits may become payable to us in respect of transactions which we arrange for you with third parties, in which case you will be notified in writing of the amount and terms of payment.
16. Investment Service
16.1 We are licensed by the Institute of Chartered Accountants in England and Wales to carry out a limited range of investment services, which are incidental to the provision of professional accountancy services and are carried out because they arise out of, or are complementary to, our provision of those accountancy services to you.
16.2 However, we are not authorised by the Financial Conduct Authority ("FCA") to carry out 'mainstream' investment business activities (as defined by the Financial Services and Markets Act 2000).
16.3 Should you require any specific investment advice, we may introduce you to an independent permitted third party ("PTP") who is authorised by the FSA and has the appropriate permissions from the FCA to undertake the regulated activity in question.
16.4 To enable us to provide you with a proper service there may be occasions when we will need to contact you without your express permission concerning investment business matters. For example it may be in your interests to sell a particular investment and we would wish to inform you of this. Therefore we may contact you in such circumstances. We shall, of course, comply with any restrictions you may wish to impose which you notify to us in writing.
16.5 In addition, we are not authorised by the FCA to carry out general insurance business. However, we are included on the register maintained by the FCA so that we can carry on insurance mediation activity which is broadly the advising on the selling and administration of insurance contracts. This part of our business, including arrangements for complaints or redress if something goes wrong, is regulated by the Institute of Chartered Accountants in England and Wales. The register can be accessed via the Financial Conduct Authority website at www.fsa.gov.uk/register/home.do
16.6 Any tax planning advice that we provide to you in the course of our engagement involving investments will consider only the possible tax consequences of changes in the portfolio. We offer no comment on the possible other merits or otherwise of particular acquisitions or disposals of specific investments, and you should seek professional advice on such matters.
16.7 Should you require us to assist in arranging any of your investment business with the PTP that PTP will take full responsibility for all aspects of compliance under any regulations required by the Act.
16.8 If we introduce you to a PTP, we will act solely as introducers and are not authorised to offer alternative advice. As a result of our introducing you to a PTP we may receive commission or other benefits from the PTP.
16.9 In the event of our receiving any such commission or benefits we will notify you in writing of the amount and terms of payment. You consent to such commissions and benefits being retained by us without our being liable to account to you for such amounts. If you would like more information on any commission payments, please contact the partner with whom you usually deal or John Coombs in our Exeter Office.
16.10 In the unlikely event that we cannot meet our liabilities to you, you may be able to claim compensation under the Chartered Accountants' Compensation Scheme.
16.11 Other investment services we may provide include the following:
• advising you on investments generally, but not recommending a particular investment or type of investment
• assisting you in making arrangements for transactions in investments in certain circumstances
• advising and assisting you on transactions concerning shares or other securities not quoted on a recognised exchange
17. Aquila Financial Management Limited
17.1 As referred to above, we may refer you to Aquila Financial Management Limited, in particular, as a specified PTP.
17.2 Aquila Financial Management Limited is an independent firm of financial advisers which has had a working relationship with Simpkins Edwards LLP for many years. Simpkins Edwards LLP are the beneficial owners of Aquila Financial Management Limited.
17.3 Aquila Financial Management Limited assumes full responsibility for the financial advice given by it, affording you the protection of the Financial Services and Markets Act 2000.
17.4 Under these arrangements we do not normally receive commission from Aquila Financial Management Limited. In the event that such commission is paid you will be notified in accordance with 14.1 above.
18. Data Protection Act 1998
18.1 Whilst the information you provide us is confidential we must observe the requirements of the Data Protection Act 1998. We may therefore process personal data such as your name and address in order to carry out your instructions. We may also pass your personal data to other professionals in order to obtain advice and to comply with our contractual obligations.
18.2 By accepting these terms and conditions you consent to such checks being made. In performing these checks personal information provided by you may be disclosed to a registered Credit Reference Agency which may keep a record of that information. This will be done to confirm your identity and a credit check will not be performed and your credit rating will be unaffected. All information provided by you will be treated securely by us and strictly in accordance with the Data Protection Act 1998.
18.3 We may use personal information provided by you in order to conduct appropriate anti-fraud checks. Personal information that you provide may be disclosed to a credit reference or fraud prevention agency, which may keep a record of that information.
18.4 In addition, we may use this information to advise you of any marketing events or provide you with information which we believe may be of interest to you. If you would prefer us not to do this please inform the person handling your matter or contact John Coombs in our Exeter office.
18.5 You have a right of access to the personal data that we hold about you. If you wish to make a Subject Access Request, please contact our Data Protection Compliance Officer, John Coombs in our Exeter Office. We are entitled to charge you a processing fee of £10 to process your request.
19. Applicable Law
19.1 These Terms of Business and the letter are governed by, and construed in accordance with, English law. The Courts of England ("Courts") will have exclusive jurisdiction in relation to any claim, dispute or difference concerning this engagement and any matter arising from it.
19.2 Each party irrevocably waives any right it may have to object to any action being brought in those Courts, to claim that the action has been brought in an inappropriate forum, or to claim that those Courts do not have jurisdiction.
19.3 If any provision in these Terms of Business or any letter of engagement, or its application, are found to be invalid, illegal or otherwise unenforceable in any respect that part of the Terms of Business or letter of engagement will be severed and the remainder shall survive unaffected to the fullest extent permitted by law.
19.4 A person who is not party to this agreement shall have no right under the Contracts (Rights of Third Parties) Act 1999 to enforce any term of this agreement. This clause does not affect any right or remedy of any person which exists or is available otherwise than pursuant to that Act.
20. Changes in the law
20.1 We will not accept responsibility if you act on advice previously given by us without first confirming with us that the advice is still valid in light of any change in the law or your circumstances.
20.2 We will accept no liability for losses arising from changes in the law or the interpretation thereof that occur after the date on which the advice is given.
21. General
We may in the future revise the terms and conditions set out in our Terms of Business and in that event such revised terms and conditions shall only apply in relation to services provided after the date of receipt of the same by you, but so that such revised terms will not affect the respective rights and obligations of the parties accrued prior to the effective date of the change.
By instructing us to commence work on this matter you will be taken to have accepted these Terms of Business.


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Italy-based Fiat S.p.A. has inked an agreement with U.S.-based Chrysler Group LLC and its shareholders to exercise an option to purchase another 16 percent stake in the second quarter for this year, the Italian carmaker disclosed on Tuesday. Under the deal, which has been faster than expected, Fiat will pay $1.27 billion for the additional stake in the U.S. carmaker.
Previously, three sources who are familiar with the matter disclosed last week that Fiat has been searching for financial sources to pay approximately $1.5 billion for the stake and would be finalized in the coming months.
The Italian carmaker can purchase the additional 16 percent after Chrysler repays government loans, giving Fiat a 51 percent control on the U.S. company.
When Chrysler emerged from bankruptcy in 2009, Fiat had an agreement with the government of the U.S. to share management and technology in exchange for a 20 percent initial stake as well as performance targets to increase to 35 percent without paying any cash.
Last week, the U.S. government gave Fiat an additional 5 percent stake in Chrysler by reaching goals that were included in Fiat executing franchise deals with 90 of its dealers in Latin America to carry Chrysler products. Chrysler is targeting to finalize its debt refinancing in the second quarter, Fiat disclosed today.


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The MCA Zine Fair is one of Australia's largest celebrations of DIY and independent art and publishing. Over the last nine years we've had hundreds of zinesters sell, trade and share their work with audiences of all ages at the fairs. Some stallholders return year after year whilst others apply for a table after visiting a previous zine fair, having been inspired to put pen to paper and share their work with the world.
In 2017, the Zine Fair returns to its original stomping ground, the MCA's Foundation Hall, for one day only. Whether you've joined us for Zine Fair before or if you're applying for the first time, here are our top tips for a great application to increase your chances at getting a coveted spot at this year's Zine Fair!
Applications close midnight, 19 February 2017. When you apply, take the time to consider your application and read through the form carefully. Even if you've had a table at a previous Zine Fair, this unfortunately doesn't automatically guarantee you a table at this year's Zine Fair. Ensuring your form is filled in, correctly and with the best samples of your work, is essential Confused about the application form? Send us a quick email at firstname.lastname@example.com and we are happy to help clarify!
Are you part of a collective? It's always great to see the variety of work presented at the fair including work from groups. What can be confusing for us as organisers is when we receive more than one application from the same group! Due to the limited number of tables at this year's fair, please choose just one member of your group to apply and provide us with a selection of the group's work when you upload your zine samples. This will avoid much confusion for everyone involved!
Just started making zines and wanting to share your work at the MCA Zine Fair for the first time? We welcome submission from bourgeoning zinesters of all ages! We acknowledge that zine making is an incredibly personal art-making experience and aim to create a fair that represents a variety of genres and styles. So don't be deterred if you've never applied before. No matter how long you have been creating, we encourage you to apply and are happy to provide feedback if your application is not successful.
Whilst many zinesters now promote and share their work through digital channels, we understand that many of you may prefer to present your work separate to the digital world. If you'd like to send samples of your work via post, don't hesitate to be in touch. Just make sure they reach us before midnight on 19 February! If you prefer to work offline entirely, please call +61 (0)2 9245 2400 to request a hard copy of the application form.
This tip is perhaps our most valuable one! When describing your zine style and philosophy, have a think about the language you use to describe your work. Whilst we endeavour to know as much as possible about the zine community in order to work with you all on this great program, we can't know everything. Provide us with a clear and concise introduction to you and your work. Are zines your entire world? You'd be surprised as to how many people will ask us 'What is a Zine?' leading up to and on the day of Zine Fair. Think about how you'd talk to those people about your work, how you can invite people to learn and discover your zines. The more we understand about your work and about you as an artist/writer/designer/photographer/skater/riot grrrl/activist/poet or however you identify as a zinester, the more chance you will have for a successful application.
Good Luck! Each year we are amazed at the many applications for the MCA Zine Fair, and we know 2017 will be no different. We can't wait to see you work!
Vivian is an arts administrator and producer who currently works at the MCA as Public Engagement Assistant. Originally from Adelaide, South Australia, she has worked in a range of arts organisations in Australia and the UK. She is interested in socially engaged and intersectional practices, performance, new media and sound-based art.


text: My name is Hannah Marquardt. I live in a small suburb of Milwaukee, called Greendale. It is there that I used to call home. My home is now at Butler University, where I am a Marketing major with a minor in Health Care Management. I thought taking a course in the College of Communications was a good idea to build on my writing skills, both professionally and non professionally. After STR128, I hope to take the skills that I have learned back to the College of Business and to continue excelling in my writing.