Sequence and expression variation in the dehydrin6 gene in barley varieties contrasting in response to drought stress

South African Journal of Botany 119 (2018) 278–285

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Sequence and expression variation in the dehydrin6 gene in barley varieties contrasting in response to drought stress S. Drine a,c,⁎,1, M. Smedley b,1, A. Ferchichi a, W. Harwood b a b c

Aridoculture and oasis cropping laboratory, Institute of Arid Regions, Medenine, Tunisia Department of crop genetics, John Innes Centre, Norwich, UK National Agronomic Institute of Tunisia, Tunis, Tunisia

a r t i c l e

i n f o

Article history: Received 5 September 2018 Accepted 19 September 2018 Available online xxxx Edited by E Balázs Keywords: Barley Drought tolerance Dehydrin6 Real-time PCR Multiple alignments of coding sequences

a b s t r a c t Dehydrins (Dhns) are the Group II Late Embryogenesis Abundant (LEA) proteins that are thought to play a key role in the response to abiotic stresses in plants. In the present study, a dehydrin6 (Dhn6) gene was isolated and characterized from eight barley genotypes (Hordeum vulgare L.) of diverse geographic origins, representing a wide range of drought tolerance. Based on analysis of physiological traits, the drought tolerance of Tunisian and Jordanian varieties and the drought sensitivity of European varieties (United Kingdom) were confirmed. To elucidate the involvement of the Dhn6 gene in the adaptive processes, its expression level was investigated under drought stress conditions. The results indicated that Tunisian and Jordanian genotypes displayed an early and rapid induction of Dhn6 expression while the UK varieties showed a delay in responding by up-regulation. Changes in expression of the Dhn6 gene, encoding a protein of unknown function, were shown to be associated with drought response mechanisms of the different genotypes. Furthermore, a multiple alignment of coding sequences of Dhn6 genes of the eight barley varieties was conducted. Two types of variation were observed in the UK varieties sequences; a deletion of 62 bp (segment ɸ) and six amino acid substitutions. Accordingly, we speculate that the observed variability in sequence and in expression of Dhn6 could be linked to the adaptability of barley to drought. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Water deficit is one of the most damaging processes affecting plants at various stages of their development (Yordanov et al. 2000). The effects of stress are often manifested at the morpho-physiological, biochemical and molecular level (Kiani et al. 2007). Therefore, several mechanisms have been adopted by plants to adapt to water stress. These include reduction in water loss by increasing stomatal resistance, accumulation of organic solutes such as amino acids and sugars (Sánchez-Díaz et al. 2008) and modifications in expression of stress responsive-genes among others (Guo et al. 2009; Hübner et al. 2015; Pandit et al. 2011). Dehydrins are the most abundant plant proteins produced in response to abiotic stresses such as drought, low temperature and high salinity (Tommasini et al. 2008). The Dehydrin family belongs to group II ofthe Late-Embryogenesis-Abundant (LEA) proteins (Close 1996). In the barley genome, 13 Dhn genes, dispersed over seven genetic map locations (Choi et al. 1999), have been reported by Rodríguez et al. (2005). These genes whichare expressed variably ⁎ Corresponding author. E-mail addresses: [email protected] (S. Drine), [email protected] (M. Smedley), [email protected] (W. Harwood). 1 Equal contribution.

https://doi.org/10.1016/j.sajb.2018.09.021 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.

during seed desiccation and in vegetative tissues when plants experience water deficiencies have recently emerged as attractive candidates for engineering of drought tolerance. The differences in expression and tissue location suggest that individual members of the Dhn multigene family have somewhat distinct biological functions (Nylander et al. 2001; Zhu et al. 2000). Linking the expression of a gene to a high degree of tolerance suggests a possible role for this gene in adaptation (Ouvrard et al. 1996). Many studies have reported a positive correlation between the accumulation of Dhns and tolerance to abiotic stresses in different species such as wheat (Lopez et al. 2003), barley (Suprunova et al. 2004), rice (Sang-Choon et al. 2005) and sunflower (Cellier et al. 1998). However, the precise function of these proteins remains unclear. Dehydrins are highly hydrophilic alkaline proteins characterized by high content of charged and polar residues. These properties allow dehydrins to interact efficiently with water and to bind a large amount of solute ions. Tompa et al. (2006) stated that this enables dehydrins to retain water and to prevent an adverse increase in ion concentration during dehydration stress. All dehydrin proteins contain the Lysine rich repeat K-segment (EKKGIMDKIKEKLPG) (Close 1996). The K-segment is predicted to form an amphipathic α-helix structure that interacts with membranes and with cellular macromolecules (Koag et al. 2009). This structure may have a chaperone-like function in stabilizing partially denatured proteins or membranes in water stress

S. Drine et al. / South African Journal of Botany 119 (2018) 278–285

environments. Several research groups have shown that dehydrins might play a crucial role as stabilizers of intracellular macromolecules during the dehydration process, thereby preserving cell structure integrity (Allagulova et al. 2003; Hanin et al. 2011; Yang et al. 2015). The lack of water in plants can also lead to accumulation of catalytic metal ions which are sources for radical generation, thereby causing oxidative damage. Hara et al. (2005) reported that dehydrin proteins may reduce the toxicity of metals by binding them and scavenging the radicals that they generate and hence may alleviate the oxidative damage of the stressed plants (Hara et al. 2004; Sun and Lin 2010). In wheat, some dehydrins accumulated in the vicinity of the plasma membrane were suggested to inhibit lipid peroxidation caused by ROS(Danyluk et al. 1998). Therefore, the accumulation of dehydrins is possibly crucial for plants to adapt to a wide range of stresses. Previous studies have shown that the over-expression of different members of dehydrin family genes enhances plant tolerance to various abiotic stresses (Liu et al. 2015; Puhakainen et al. 2004; Xing et al. 2011). Thus, there is a need to add new insight to the molecular aspects of drought tolerance in barley and to clarify the role of single members of the DHN gene family in the general pattern of water stress response. The objectives of the current study were therefore: to investigate the sequence and expression variations of the Dhn6 gene, in seedlings of barley varieties contrasting in response to drought stress and to pave the way for future work to clarify the role of the Dhn6 gene in drought tolerance by genetic engineering. 2. Materials and methods 2.1. Plant materials Eight barley varieties from North Africa (Tunisia), Middle East (Jordan) and Europe (UK) were used throughout this study. These varieties were selected by visual scoring of drought related secondary traits and yield characteristics under field conditions. These apparently sensitive and resistant genotypes had previously been analyzed for physiological parameters (proline, malondialdehyde (MDA) and relative water contents) after exposure to short-term drought stress at the seedling stage (Drine et al. 2017). Seeds of Jordanian barley varieties: Rum (Harbin-Arivat x Attiki CYB191A-0A-0A-0A), Mutah (Roho-A. Abiad-6250) and UK varieties: Maythorpe (Maja x Irish Goldthorpe), Golden Promise (mutant of Maythorpe), Oxbridge (Tavern x Chime) and NFC-Tipple ((NFC 497-12 x Cork) x Vortex) were obtained from the John Innes Centre Germplasm Resources Unit (Norwich, UK). The seeds of barley cultivar Martin (Unknown Algerian population) were kindly supplied by Pr M. El Falah (INRAT, Tunisia) and Ardhaoui, the unique local barley cultivar of the south of Tunisia, was obtained from the Arid Regions Institute (IRA, Tunisia). 2.2. Experimental design Barley seeds were sterilized by firstly washing in 70% ethanol for 30 s followed by three washes in sterile distilled water and then in a solution of sodium hypochlorite diluted 50:50 with water for 4 min and thoroughly rinsed with distilled water. Sterile seeds were germinated on moist filter paper inside Petri dishes, and then incubated at 22 °C with 16 light/8 h dark photo cycle, for 5 days. Seedlings were gently removed from the filter paper when roots were sufficiently developed. Barley seedlings of a similar developmental stage were transplanted in hydroponic culture in half-strength Hoagland's solution (Hoagland and Arnon 1950) with doubled iron concentration. Solutions were aerated by constant aeration using commercially available aquarium pumps. During hydroponics experiments, seedlings were grown in a controlled environment room (CER) at 20 °C day and 15 °C night temperatures, 75% relative humidity, and photoperiod of 16/8 h (day/night) with light levels of 500 μmol m−2 s−1 (provided by metal halide bulbs (HQI) supplemented with tungsten bulbs).

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Two-week-old seedlings designated for drought treatment were transferred to half-strength Hoagland's solution containing 20% (w/v) of polyethylene glycol (PEG6000) (corresponding to −0.6 Mpa water stress) whilst control seedlings were transferred to fresh Hoagland's solution(×0.5). Whole leaf tissue from each individual plant was harvested at 0; 6; 12; 24 and 48 h of drought stress, flash frozen in liquid nitrogen, and then stored at −80 °C prior to RNA extraction. 2.3. mRNA isolation and qRT-PCR Total RNA was extracted from 100 mg of leaf samples using a QIAGEN RNeasy Plant Mini kit. The RNA was further purified using the QIAGENRNase-Free DNase set to ensure removal of genomic DNA contamination. RNA quantity and quality was assessed using the NanodropND-1000 spectrophotometer (Thermo Fisher) and agarose gel electrophoresis. For cDNA synthesis, one microgram of RNA was combined with 2.5 μl of 10 μM Oligo(dT)18- VN(5′-TTTTTTTTTTTTTTTTTTVN-3′), and the volume was made up to 14 μl with H2O. This reaction mixture was incubated at 72 °C for 5 min, and then transferred straight onto ice. One microlitre of M-MLV reverse transcriptase (Invitrogen), 5 μl of 5X reverse-transcriptase reaction buffer, 2.5 μl of 0.1 M DTT (Invitrogen) and 2.5 μl of 10 mM dNTPs were added. The reaction was then incubated at 37 °C for 75 min, followed by 70 °C for 15 min and 4 °C for 1 min. Gene quantification was performed using quantitative reversetranscriptionPCR that was carried out in singleplex using SYBR Green. Three independent seedling samples for each genotype were analyzed in triplicate. Reactions were prepared in 96-well plates to a final volume of 20 μl; with 2 μl diluted cDNA (1:4), 10 μl of SYBR Green JumpStart™ Taq ReadyMix (Sigma-Aldrich), 0.8 μl of primer mix (containing forward and reverse primers at a concentration of 10 μM) and 7.2 μl of nuclease-free water. Thermal cycling conditions consisted of 44 cycles of 95 °C for 10s, 60 °C for 10s and 72 °C for 20s, plus a final extension at 72 °C for 2 min. The primers Dhn6F (5′-TTTTACCGTGTGATAGATGTTGCA-3′) and Dhn6R (5′-TGCAAACCGACCAGACAAACT-3′) were used to amplify a 72bp portion of the Dhn6 gene (accession number AF043091) (Suprunova et al. 2004).A gene encodingUbiquitin (accession number M60175.1) from barley (Rapacz et al. 2012), was used as internal control and this gene was amplified with the primer pair (Forward: 5′-TCGCCGTCCTCCAGTTCTAC-3′; Reverse: 5′-CCTTCCTGAGCCTGGTTAC CT-3′) which produced an amplicon of 63 bp. These primers were evaluated for PCR amplification efficiencies by performing real-timePCR and using serial dilutions of cDNA sample that showed the maximal amount of target gene, at rates of 1, 1/10, 1/ 20, 1/50, and 1/100. For each primer pair, the efficiency assay revealed a standard curve with high linearity (R2 N 0.98) and consistency among replicates. The target and reference genes have similar and nearly 100% amplification efficiencies. The Δ CT method, a variation of the Livak method (Livak and Schmittgen 2001), was used for calculating relative gene expression for each sample with the formula to the power of _[mean CT(target)– mean CT(reference)]. The obtained data was subjected to one-way analysis of variance (ANOVA). The mean values were compared using Duncan's multiple range tests at P = 0.05. XLSTAT 2014 software was used to carry out statistical analysis. 2.4. Cloning and sequencing A modified version of the Edwards et al. (1991) protocol was used to extracttotal genomic DNA from 6-day-old fresh seedlings ofbarley varieties.

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Partial amplification of the Dhn6 gene was used for sequencing using these oligonucleotide primers: GapF 5′-ACCGGTACCCACGGAACT-3′ and GapR 5′-GCTTGTGTACCGAGCTGGAG-3′. Based on the Hordeum vulgare Dhn6 sequence (AF043091) (Choi et al. 1999), primers were designed to amplify a 679-bp fragment in the coding region of Dhn6 gene. PCRefficiency was optimized by selecting the most efficient temperature from a gradient. PCR reactions were performed in a 25 μl volume containing 50 ng of genomic DNA, 200 μM dNTPs, 0.5 μM of each primer (Gap F and Gap R), 1.25 U Pfu Ultra High-FidelityDNA polymerase (Agilent Technologies) and 1× PCR buffer. PCR conditions were 95 °C for 2 min; then 37 cycles of 95 °C for 30s, 60 °C for 40s, 72 °C for 38 s; followed by a final extension of 72 °C for 7 min.The expected product should be 679 bp.The success of the amplification and the size of the Dhn6 product were assessed by electrophoresis and comparison against a 1Kb plus DNA ladder (Invitrogen). PCR products were purified with QIA quick PCR Purification Kit (Qiagen), A-tailed as described in Smedley and Harwood (2015) and cloned into pGEM-T Easy vector system (Promega) following the manufacturer protocol. Transformed cells were grown with shaking (220 rpm) in overnight culture (10 ml LB,50 μg/ml carbenicillin) at 37 °C, before plasmids were isolated from Escherichia coli (Library Efficiency® DH5α™ cells, Invitrogen) using QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturer's instructions. In order to confirm the presence of the insert, a restriction digest was performed in a 10 μl volume containing 1 μl restriction enzyme EcoRI (10 U/μl), 1.5 μl plasmid DNA and 1 μl buffer (10 ×)and then incubated at 37 °C for 75 min. Gel electrophoresis was used to analyze plasmid DNA digests. Positive recombinant plasmids that contained the Dhn6 insert were sequenced using the M13 forward and reverse sequencing primers. Sequencing was performed by The Genome Analysis Centre (TGAC, Norwich, UK) and sequences were aligned using AlignX, from Vector NTI Suite1 (Invitrogen). 3. Results On the basis of the results reported by Drine et al. (2017), the examined barley genotypes were different in their responses to short term stress at the seedling stage, according to the measurements of RWC, proline and MDA contents. Tolerance to drought stress of the Tunisian and Jordanian barley cultivars was associated with significant increase in proline concentration, with little change in MDA content and smaller decrease in leaf water content compared to UK varieties. The differences in physiological responses classified these genotypes in two groups based on the Euclidean distances. The susceptible varieties are gathered in the same cluster recording an average Euclidean distance of 27.2 and an average dissimilarity of 52.4 compared to the drought-tolerant group.

As a result of this screening, Tunisian and Jordanian barley genotypes were defined as more tolerant to drought stress compared to UK varieties. To clarify the role of Dhn6 gene in the adaptive processes, its expression level was examined under drought stress conditions. 3.1. Quantitative real-timePCR analysis of Dhn6 gene expression To determine the expression of Dhn6, a member of the dehydrin gene family, Q-PCR was carried out with cDNA obtained from the eight barley genotypes after 6, 12, 24 and 48 h of PEG stress treatment. The expression of the reference gene ‘Ubiquitin’ among eight genotypes was similar under control and drought stress conditions. The results reported by Rapacz et al. (2012) indicated that ‘Ubiquitin’ is the most suitable reference gene to study drought-induced changes in gene expression at the barley seedling stage. In non-stressed seedlings, Dhn6 gene expression was almost undetectable, while differences in expression pattern were observed between the eight genotypes under drought stress as can be seen in Fig. 1. All the examined genotypes subjected to 6 h of drought stress showed a slight increase in the expression level of Dhn6. After 12 h of PEG treatment, significant differences in transcript level of Dhn6 were observed in the eight barley genotypes (P b .001). At this time point of drought treatment, the accumulated levels of Dhn6 transcript were higher (more than three fold) in Tunisian and Jordanian genotypes than in the UK varieties with the genotype Rum displaying the highest expression level. After 24 h of drought stress the UK varieties showed an increase in their Dhn6 expression levels. The Tunisian and Jordanian genotypes reached their maximal transcript accumulations after 24 h of PEG treatment, while the UK varieties showed their maximal levels after 48 h of drought stress. The expression of Dhn6 was not changed in Martin and increased slightly in Rum after 48 h of PEG stress, however a decrease in Dhn6 expression levels was observed in Ardhaoui and Mutah genotypes. After 48 h of PEG treatment the eight barley genotypes exhibited almost similar levels of transcript accumulation (P = 0.018) and the highest transcript level was detected in the Golden Promise variety. 3.2. Cloning and sequence analyses of Dhn6 fragment The Dhn6PCR amplicon, consisting of 679bp of open reading frame (ORF) sequence, was amplified using the Gap forward and Gap reverse primers as described in Section 2.4. The size of the amplified products obtained from UK varieties was estimated from electrophoresis to be about 650 bp. However, bands of around 700 bp in size were observed when the PCR products obtained from Tunisian and Jordanian genotypes were analyzed electrophoretically (Fig. 2).

1.2 1 0.8

6H 12H

0.6

24H 0.4

48H

0.2 0 Ar

Mr

Mu

Rm

Gp

My

Tp

Ox

Fig. 1. Relative levels of Dhn6 expression of the reported genotypes. Q-PCR was carried out with mRNA isolated from control and PEG-stressed barley seedlings after 6, 12, 24 and 48 h. Quantification is based on Ct values that were normalized using the Ct value corresponding to barley Ubiquitin gene. Three independent seedling samples for each genotype (Ar: Ardhaoui, Mr.: Martin, Mu: Mutah, Rm: Rum, Gp: Golden promise, My: Maythorpe, Tp: Tipple, Ox: Oxbridge) were examined in triplicate. Vertical bars represent the standard error of the mean values.

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1

2

3

4

M

5

6

7

8

N

281

M

3000 bp 2000 bp 1650 bp 1000 bp 850 bp 650 bp 500 bp 400 bp

Fig. 2. Agarose gel electrophoresis of the amplified product of Dhn6. The amplified PCR products were separated in 1% agarose gel (TBE buffer) stained with ethidium bromide. Lane 1–4 (1: Golden promise, 2: Maythorpe, 3: Tipple, 4: Oxbridge); laneM: molecular weight marker 1Kb plus DNA ladder (Invitrogen); lane5–8 (5: Ardhaoui, 6: Martin, 7: Mutah, 8: Rum); lane N: negative control.

Following cloning of the PCR products, positive recombinant plasmids that contained the Dhn6 insert were sequenced. Analysis of the DNA sequences of the different varieties revealed the presence of point substitutions as well as larger deletions. The alignment of coding nucleic acid sequences of Dhn6 from the studied varieties revealed a 62 bp deletion at +364 bp in UK varieties (Fig. 3). Moreover, six point substitution mutations (which substitute one base for another) were also detected at + 87 bp, + 321 bp, +322 bp, +432 bp, +498 bp and + 539 bp (Fig. 3). Compared with UK varieties, there were two small deletions in the Jordanian and Tunisian varieties, 6 bp at +99 bp (lacking TCCACC), as well as 9 bp at +110 bp (lacking AGGGATCAC) (Fig. 3). The full-length coding region of the Dhn6 gene from the Hordeum vulgare cultivar ‘Ardahoui’ has been deposited in the NCBI database under GenBank accession number HQ609471.1. Its structure is shown schematically in Fig. 4. The position of the deletion of 62 bases in the Dhn6 coding region of the United Kingdom varieties is shown in Fig. 4. The analyses of deduced amino acid sequence of Dhn6 show that this protein (Y2SK3) is characterized by conserved sequence motifs designated K,S and Y and by less conserved regions, usually rich in polar amino acids (the Phi-segments) (Fig. 4). 4. Discussion The development of drought-resistant crop varieties is an important strategy to meet global food demands with less water. However, this requires understanding of the adaptive mechanisms deployed during a water deficit. Dehydrins represent one of the most analyzed drought-inducible gene families, which are involved in plant protective reactions against the damage caused by dehydration (Close 1997; Zhu et al. 2000). Drought tolerance is a complex trait controlled by a number of quantitative trait loci (QTLs). In fact, several QTLs controlling drought tolerance traits have been identified close to dehydrin genes that may be key genetic determinants of drought tolerance. The Dhn6 gene is located on barley chromosome 4(4H) (Choi et al. 1999). According to WójcikJagła et al. (2013), Ma et al. (2012) and to Molnár et al. (2007), genes mapping to chromosome 4H appear to be especially important for adaptation to drought affected environments. The HvDhn6gene encoding a LEA class2 protein, was silenced in barley by using VIGS (Virus-induced gene silencing) (Liang et al. 2011). Under drought stress, Dhn6-silenced plants showed lower survival rates compared to control plants. These results suggested the role of Dhn6 in the adaptive response of barley to water deficit. In order to understand the relationship between phenotypic adaptations and molecular characteristics of dehydrins, we cloned Dhn6 genes from barley varieties contrasting in response to drought stress according to Drine et al. (2017).

Analysis of nucleotide sequences found that there was specific variation in this gene. Two types of variations were identified in UK varieties, sensitive to water stress, compared to drought tolerant varieties: a deletion of one segment Φ (rich in glycine and polar amino acids) and six point substitutions. These types of variations are similar to those observed, between the Dhn alleles of barley, by Choi et al. (1999) and Yang et al. (2012), who found that the most common allelic variations involve deletions or duplications of Φ segments. Such variations in UK varieties may affect the level of expression of this gene. The genetic divergence of amino acid sequences between wild barley populations from Africa and Europe, observed by Yang et al. (2012), suggested that Dhn6 has been subjected to the natural and adaptive selection associated with drought resistance. Therefore, it was interesting to study the expression of the Dhn6 gene to reveal the relationship between the genetic divergences of the sequences observed between the Tunisian, Jordanian and the United Kingdom genotypes and the tolerance to water stress. In our study, a variation in gene expression among the different barley genotypes was shown during drought stress induced by PEG6000 in hydroponic conditions. Our results showed a rapid increase in the transcript level of Dhn6 in Tunisian and Jordanian genotypes after 12 h of drought stress, while the UK varieties showed a delay in responding by up-regulation. Our findings are in line with those of Qian et al. (2008) and Suprunova et al. (2004) on hull-less and wild barley genotypes subjected to dehydration treatment. These authors reported that the higher and earlier up-regulation of the Dhn6 gene was associated with drought tolerant genotypes. According to Yang et al. (2012), the expression of the Dhn6 gene is strictly related to drought in barley. Qian et al. (2008) noted that the Dhn6 gene was expressed in drought resistant genotypes after 8 h of dehydration treatment, while the earlier expression of Dhn6 in the Tunisian and Jordanian genotypes was revealed after 12 h of drought stress induced by PEG. The expression of most dehydrin genes was found to be dependent on both the genotype andthe severity of water deficiency (Rampino et al. 2006). Themaximum expression level of peudhn1 in Populus induced by PEG 6000 in hydroponic conditions was lower than the effect induced by withholding water, but higher than those obtained in cuttings growing in pots and treated with PEG 6000 or NaCl (Caruso et al. 2002). In fact, the addition of PEG 6000 or NaCl to soil slowly increased the stress and resulted in a progressive decrease of the predawn leaf water potential, a decrease of stomatal conductance and a growth reduction associated with a progressive increase of dehydrin transcript level. However, treatment with PEG 6000 in hydroponic conditions caused a faster reduction of stomatal conductance associated with a rapid induction of dehydrin. Tunisian and Jordanian genotypes displayed an early and rapid induction of Dhn6 expression. Based on the analysis of physiological traits (Drine et al. 2017) and Dhn6 transcript accumulation, Tunisian and Jordanian genotypes showed a higher ability to adapt to the stress

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Fig. 3. Alignment of coding nucleic acid sequences of Dhn6 from the eight barley varieties (Gp: Golden promise, My: Maythorpe, Ox: Oxbridge, Tp: Tipple, Ar: Ardhaoui, Mr.: Martin, Rm: Rum, Mu: Mutah). Identical residues are highlighted in yellow and non-identical ones are shaded in blue. Single-nucleotide polymorphisms are highlighted in green. Dashes “-”indicate gaps in a sequence. The sequences were aligned using Vector NTI Suite 10.

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Fig. 3 (continued).

condition imposed. Yang et al. (2012) suggested that the observed differences of Dhn6 expression in barley, originating from different micro- and macro-ecogeographic locations, may be the result of adaptive edaphic and climatic selective pressures. Suprunova et al. (2004) hypothesized that resistance to drought stress could be linked to different mechanisms such as the high expression level of dehydrin genes, the presence of more efficient transcriptional activators and the early perception of the stress. de Mezer et al. (2014) indicated that induction of the dehydrin genes in response to a progressive increase in water deficit is more associated with the rate of water loss from barley leaf tissues than their ability to adapt to water deficit. These results were not consistent with those previously reported by Rampino et al. (2006) indicating that under drought stress in resistant wheat genotypes, the induction of dehydrin genes is triggered even though tissue hydration levels (RWCs) are still high, also indicating the involvement of these proteins in water retention. These results agree well with our findings in the present study. In the drought-tolerant cultivar Ardhaoui, induction of the Dhn6 gene was activated after 12 h of drought stress despite the fact that the RWC level of this genotype was still high (85%) after 72 h of drought stress. At the same time,

less damage to the cell membranes and a high proline accumulation were observed in this genotype (Drine et al. 2017). The early and high expression levels of Dhn6 were closely related to the tolerance of Tunisian and Jordanian barley cultivars and therefore might be valuable in plant stress resistance evaluation. Furthermore, tolerance to drought stress in our studies was accompanied by more slowly changing ratios of MDA content suggesting a higher oxidative scavenging ability in the drought tolerant genotypes (Drine et al. 2017). This could be due to the roles of dehydrins, in eliminating ROS and protecting the plasma membranes from damage by environmental stresses. Dehydrins, such as CuCOR19, were proposed to directly scavenge radicals and decrease the oxidative damage induced by water stress (Hara et al. 2004). Plant dehydrins are suggested to be ion sequestering anti-oxidative proteins which can prevent the metal requiring production of radicals. This is mainly due to their amino acid composition. The content of amino acid residues which are implicated in metal–protein interactions are higher in dehydrins in general (Battaglia et al. 2008). The protein encoded by the Dhn6 gene contains the highest proportions of Gly (32.5%) and His (9.2%) compared to the other barley dehydrins (Sun and Lin 2010). Therefore, we can suggest

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50 bp repeat 43/50 bp repeat repeats are f-segments - Gly/Thr rich

Gap in GP, Maythourpe, Oxbridge and Tipple 50 bp repeat

49/50 bp repeat

42/50 bp repeat

44/50 bp repeat

27/29 of the 50 bp repeat

31/35 of the 50 bp repeat

S segment undergoes phosphorylation

49/50 bp repeat

Gap reverse primer Intron

44/50 bp repeat Gap forward primer

S segment continued after intron

Y2 nucleotide-binding site

K1 Lysine-rich repeat amphipathic helix

Y1 nucleotide-binding site

K3 K2

start

Stop

Dhn 6 1607 bp Fig. 4. Structure of Dhn6 Open reading frame sequence from Hordeum vulgare cultivar Ardahoui and the 62-bp gap position in Golden promise, Maythorpe, Oxbridge and Tipple. The Dhn6 PCR amplicon, consisting of 679 bp of the ORF sequence, was amplified using Gap forward and Gap reverse primers (indicated in the schema). The big green arrow is marking the Phi-segment repeats.

its possible involvement in the anti-oxidative system and consequently in drought tolerance. Our findings do indicate clear differences in expression profiles of Dhn6 between drought tolerant and more susceptible cultivars and clear genomic variation specifically associated with drought tolerant cultivars. This work suggests some intriguing possibilities for engineering transcription factors, such as Dhn6, possibly using genome editing technologies, to improve drought tolerance in European varieties.

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