Molecular Characterization of Dehydrin in Azraq Saltbush among Related Atriplex Species

Atriplex spp. (saltbush) is known to survive extremely harsh environmental stresses such as salinity and drought. It mitigates such conditions based on specialized physiological and biochemical characteristics. Dehydrin genes (DHNs) are considered major players in this adaptation. In this study, a novel DHN gene from Azrak (Jordan) saltbush was characterized along with other Atriplex species from diverse habitats. Intronless DHN-expressed sequence tags (495–761 bp) were successfully cloned and sequenced. Saltbush dehydrins contain one S-segment followed by three K-segments: an arrangement called SK3-type. Two substantial insertions were detected including three copies of the K2-segemnet in A. canescens. New motif variants other than the six-serine standard were evident in the S-segment. AhaDHN1 (A. halimus) has a cysteine residue (SSCSSS), while AgaDHN1 (A. gardneri var. utahensis) has an isoleucine residue (SISSSS). In contrast to the conserved K1-segment, both the K2- and K3-segment showed several substitutions, particularly in AnuDHN1 (A. nummularia). In addition, a parsimony phylogenetic tree based on homologs from related genera was constructed. The phylogenetic tree resolved DHNs for all of the investigated Atriplex species in a superclade with an 85% bootstrap value. Nonetheless, the DHN isolated from Azraq saltbush was uniquely subclustred with a related genera Halimione portulacoides. The characterized DHNs revealed tremendous diversification among the Atriplex species, which opens a new venue for their functional analysis.

Many efforts have been directed toward the characterization of relevant salt-responsive genes underlying the unique physiology of halophyte plants [12,13]. In this regard, saltbush would sense salinity stress or even drought stress through a specialized protein, which was previously called early responsive to dehydration (ERD). The new name for ERD is the osmosensitive calcium-permeable cation channel (OSCA). The OSCA was first reported in Arabidopsis by two separate research groups [14,15]. In saltbush, AhOSCA expression was found to be upregulated ca. 6-folds in A. halimus under 150 mM NaCl level (ca. 13 dS m −1 ), which mimics the salinity levels in the Azraq region [2].
Dehydrins (DHNs) are widely spread across the plant kingdom and are considered crucial stress-responsive genes, e.g., FcDHN in common fig under salinity stress [9] and tomato TAS14 dehydrin under drought stress [16]. DHNs contain hydrophilic residues that have functional changes in response to solutes and dehydration [13,17]. DHNs are members of the Late Embryogenesis Abundant II (LEA II) protein family, which are considered stressresponsive factors and are predominant during seed maturation and dissection phases [17]; in addition, they are considered to be major salinity tolerance biomarkers [13]. Nonetheless, there are other important salinity biomarkers, and some overlap even with drought stress but others do not [16].
DHN has three conserved segments in its structure (Y, S, and K segments), where K segments can be available once, twice, or trice forming amphipathic α-helices, which aid in stabilizing cellular components and membranes [18]. Both α-amylase and lactate dehydrogenase are examples of cold-sensitive enzymes protected by DHNs [19]. Additionally, DHNs can protect against oxidative stress [20]. Different Atriplex species are widely distributed around the world with diverse prevailing environmental conditions. Therefore, a colinearity is expected between the protein structure and function for each species, which would be vital for adaptation. Therefore, this study aimed to characterize DHN from Azrak (Jordan) saltbush among a group of diverse Atriplex species at the molecular level.

Plant Materials
The saltbush seeds were collected from Azrak (Jordan). In addition, seeds from different Atriplex spp. were kindly provided from two seed banks: the National Agricultural Research Center (NARC, Jordan) and the National Arid Land Plant Genetic Resource Unit (USDA, USA). The seeds were surface-sterilized and germinated in vitro over MS medium. The plantlets were subcultured using nodal cutting [13]. A total of fourteen accessions covering nine different species were included in the study (Table 1): thirteen Atriplex spp. accessions and Halimione portulacoides (sea purslane), a sister species that was formerly classified as A. portulacoides.

Cloning DHNs
Total RNA was extracted from fresh leaves based on the guanidinium thiocyanate phenol-chloroform method [20] using TRIZOL (Invitrogen Inc, USA). All used plasticwares were RNase free, and RNA was resuspended in DEPC-treated water (Qiagen, Germany) supplemented with an RNase inhibitor (Qiagen, Germany). Reverse transcription reactions were performed for isolated RNA templates following the recommended procedure using a GoScript™ Reverse Transcriptase kit (Promega, USA). Several primers were designed to amplify ESTs covering the majority of DHN genes using the polymerase chain reaction (online Supplementary Table S1). The fragments were cloned as described earlier [13] and sequenced with the Sanger method using an ABI 3730 sequencer (ABI, USA). The sequences were deposited in GenBank [21] with nucleotide accession numbers (MH591427-MH591440) and corresponding protein accession numbers (AYH52682-AYH52695) ( Table 1).

Analysis of Atriplex DHNs
Protein secondary structure was predicted using Phyre2 software [22]. Protein features and predicted 3-dimensional structure were analyzed through the PSIPRED server hosted by University College London [23]. The three-dimensional structure with ligand prediction was predicted using I-TASSER software [24]. DHN proteins from related Chenopodiaceae were retrieved from Genbank [21]. They included proteins from two Atriplex species, Chenopodium quinoa, Beta vulgaris, Spinacea oleracea, and two Suaeda species (salsa and glauca) in addition to Tamarix hispidata as an outgroup. Protein sequences were subjected to multiple sequence alignment, along with saltbush dehydrin protein sequences generated in this study using BioEdit [25]. Aligned sequences were bootstrapped 1000 times by using the SEQBOOT function available in PHYLIP software [26] followed by the construction of a phylogenetic tree based on the parsimony method. A consensus tree was illustrated using TreeView [27].

Results
Seedlings were successfully grown for all Atriplex species. Thereafter, nodal cuttings were used to multiply and maintain the cultures in 250 glass bottles to obtain enough tissue materials for RNA isolation. The sequenced DHN ESTs were found to be in the range of 495-761 bp. Nonetheless, they cover all important segments (S and K).
When the DHN amino acid sequences were multiple-aligned, two major insertions were detected. The first insertion was short (KHETLGQ) and was detected in DHN protein from A. canescens (AFC98463), which was located 14 amino acids upstream of the S-segment. The second insertion detected 9 amino acids upstream of the K2-segment. Two different segments in the second insertion were evident; the first segment (Fifty amino acids long) has an additional K2-segment and was detected in A. halimus (AGZ86543), A. dimorphostegia (AYH52684), A. leucoclada (AYH52687), A. hortensisi (AYH52688), and A. gardneri var. utahensis (AYH52691). Interestingly, A. numimularia (AYH52689) showed a similar fifty amino acid insertion but without an additional K2-segment as above. The second segment (one hundred amino acids long) has two extra K2-segments, and it was detected only in A. canescens (AFC98463).
A membrane pore-lining stretch (14-6 amino acids) was predicted for all of the investigated DHNs (online Supplementary Table S2), where the N-and C-termini were cytosolic and extracellular, respectively, except for A. nummularia, which had two DHNs; AYH52689 had an extracellular N-terminus and a pore-lining stretch of EDAVISGVEKAHVFS (Figure 2A), while AYH52690 had a cytosolic N-terminus and a pore-lining stretch of HEAVT HVATAEPSVEG ( Figure 2B). The secondary structure prediction for the investigated Atriplex DHNs was carried out for polypeptides spanning [N-terminus-S-segment-K1segment] (online Supplementary Figure S1). Accordingly, Atriplex DHNs showed unique helices with diverse numbers and lengths. Concerning the conserved segments in DHN proteins, most species showed six residues of the conserved S-segment (SSSSSS) ( Figure 3). However, A. gardneri var. utahensis (AYH52691) has the isoleucine (I) residue at the second position, and A. nummularia (AYH52690) has the threonine (T) residue at the second position. Moreover, A. nummularia (AYH52689) showed three cysteine (CCC) residues, and A. halimus (AYH52686) showed one cysteine residue (C) at the third position ( Figure 3). Analysis of aligned DNA sequences showed several SNPs in the S-segment (S1S2S3S4S5S6) for the investigated Atriplex species. In S1, a silent mutation (TCC to TCT) was found in three A. halimus accessions (KF578414, MH591427, and MH591431). A S2I missense mutation was found in A. gardneri var. utahen-sis (AYH52691) as a result of a codon change from the consensus AGC to ATC. In addition, the A. nummularia (AYH52690) showed a S2T variant due to a codon change from the consensus AGC to ACC. Moreover, sequential serine to cysteine substitutions (S2C, S3C, and S4C) were detected in A. nummularia (AYH52689) due to three SNPs in the consensus (AGC TCT AGC to TGC TGT TGT). In addition, the A. halimus (AYH52686) DHN showed a S3C variant due to a codon change from the consensus TCT to TGT. The highly conserved K1-segment (KKKRKKEKKEKK) was evident for different Atriplex species and accessions ( Figure 3). Three amino acid substitutions were found in A. canescens (AFC98463); K3R was due to a codon change from AAG > AGG; K5R and K8R, the last two substitutions, resulted from a codon change from AAG to AGG.
The K2-segment is the third major motif present in DHN proteins with a consensus of KKGGFLDK(V/I)KDK ( Figure 3). However, it was KKGGFVEKIKDK in A. canscence (AFC98463), while it was KKVGFLDKVKDK in several species; A. halimus (AYH52685), The three-dimensional structure was predicated for the DHN protein from A. halimus (AYH52683) grown in the Azraq region (Jordan). The structure contains eight long and three short helices ( Figure 4). Three ligands were predicted to bind the protein, namely, Mg, Ca, and Fe. A parsimonious phylogenetic tree was constructed for all available DHN proteins from the Atriplex species and the homologs from other species in the Chenopodiaceae ( Figure 5). The Tamarix hispidata (out-group) DHN was nicely separated from all of the other DHNs. The tree showed two major clusters. The first one was resolved with a 98% bootstrap value. It comprises two DHNs from Chenopodium quinoa.

Discussion
We were able to amplify DHN genes from several Atriplex species both indirectly using cDNA and directly using genomic DNA as they were found to be intronless. Likewise, several DHNs from different plant species were found to be intronless, e.g., Eucalyptus globulus [28] and Vigna radiate [29]. Many plants' stress-responsive genes are void of introns or interspaced with just a few ones, e.g., MYB transcription factor [30] and salinity-responsive genes [31]. Furthermore, stress-responsive genes with rapid change in expression levels were also found to be interspaced with limited introns, e.g., Arabidopsis thaliana [32]. Moreover, recent studies have shown that such intronless and limited intron-interrupted genes were putatively acquired by horizontal gene transfer from prokaryotes to Phragmoplastophyta long before the appearance of terrestrial plants [33,34].
We were interested in investigating DHN genes from saltbush from Azraq (Jordan) and different Atriplex species as they were directly involved in salinity tolerance [16,35]. This was achieved by utilizing available sequences for DHN genes: A. halimus and A. canscence both with complete ORF. A recombinant yeast-expressing AcDHN gene was found to tolerate salinity stress [35]. Likewise, the expression of the AhDHN gene was found to be upregulated by more than seven folds in A. halimus roots under salinity stress [13]. However, they code for proteins that vary in structure and size. While the AhDHN gene (KF578414) from Saudi Arabia isolate encodes a 26.8 kDa protein [13], the AcDHN gene (JN974246) encodes a 38.3 kDa protein [35]. When compared with orthologs from the related genus Chenopodium quinoa, a wider range in protein sizes is evident, i.e., 30,34,50, and 55 kDa [36].
DHNs vary in their cellular location (membrane or cytoplasmic). They can have multiple locations in the cell, e.g., wheat DHNs [37]. Nonetheless, all of the investigated Atriplex DHNs in this study showed a single predicted pore-lining (online Supplementary Table S2) in comparison to comparable stress-responsive proteins with two pore-linings, e.g., OePMP3 [10]. Most Atriplex DHNs showed an extracellular C-terminus and a cytoplasmic N-terminus, similar to the previously published AhDHN [16]. However, Atriplex nummularia has two DHNs; one has an extracellular C-terminus and a cytoplasmic N-terminus similar to the other investigated DHNs, while the other has an extracellular N-terminus and a cytoplasmic C-terminus. The putative pore-lining of Atriplex DHNs indicates that they have major hydrophobic domains and consequently facilitate the folding of this integral protein along the phospholipid bilayer membrane. Likewise, plant DHN homologs were found to be associated with cell membranes. Arabidopsis DHNs were found to bind aquaporin (AtPIP2B) indicating a potential role in maintaining the lipid association of the aquaporin hydrophobic transmembrane portion [38]. Moreover, some plant DHNs were found to bind abiotic stress-responsive proteins for protection and to enhance their activity, e.g., ERD14 which can bind to Phi9 GLUTATHIONE-S-TRANSFERASE9 and CATALASE under oxidative stress in Arabidopsis [39], GsPM30 interacting with receptors such as cytoplasmic kinase GsCBRLK under salinity in wild soybean [40], and MtCAS31 protecting leghemoglobin under drought stress in barrel medic [41].
The detected amino acid substitutions in the major segments in the A. halimus DHN (Azraq region, Jordan) would interfere with its interaction with other macromolecules. For example, it was found that the S-segment is a major phosphorylation domain in Arabidopsis DHN [42]. Moreover, K-segments are positively charged as they are rich in K residues; therefore, they can bind DNA as the phosphate backbone is negatively charged [43]. Moreover, the expression of saltbush DHN (Azraq region, Jordan) was assessed using qRT-PCR (data not shown). The results were almost comparable with earlier findings for DHN expression under salinity stress (Al-Jouf region, Saudi Arabia) [13]. The saltbush DHN (Azraq region, Jordan) showed around ten-fold upregulation in the root tissues rather than the shoots compared with the seven-fold upregulation in DHN (Al-Jouf region, Saudi Arabia) [13].
Two GO terms related to molecular function were revealed for A. halimus DHN (Azraq region, Jordan), namely "substrate-specific transporter activity" and "protein binding", while it showed four GO terms related to biological processes, "protein localization to nucleus", "nuclear import", protein import", and "protein targeting". Likewise, the maize DHN was found localized to the nucleus [44]. In addition, "nuclear envelope" and "pore complex" were predicted as GO terms related to cellular localization. In fact, both H residues and K-segments are indispensable for binding phospholipids, a major component of the cellular membrane [45,46].
On the other hand, three metal-ligand binding sites were predicted in the DHN from A. halimus (Azraq region, Jordan) ( Figure 4). The first was a Mg ligand binding site with residues D148 and D152. The second was a Ca ligand binding site with residues D148 and D152. Similarly, the Arabidopsis DHN was found to bind calcium, which was found to be directly proportional to phosphorylation [42]. Finally, there was a Fe ligand binding site, which was associated with residues D162 and K191. A similar affinity to Fe was recorded in Vitis riparia DHN1 [47]. Furthermore, DHNs binding metal ligands can act as reactive oxygen species scavengers, which would aid in plant mitigation under various stresses [43,45,46].
On the contrary, in a previous study, A. halimus was clustered away from A. dimorphostegia based on the atpB-rbcL spacer sequence, while A. nummularia and A. hortensis were clustered together and away from A. halimus and A. leucoclada based on ITS sequences [48]. This could mean that the DHN protein sequence is more conserved among Atriplex spp than DNA-based sequences (e.g., the atpB-rbcL spacer and ITS sequences). Atriplex and Chenapodium were clustered together but away from Beta vulgaris (Subfamily Betoidieae) based on trnL-F and rpl16 sequences [49], the ITS sequence [50], and the matK/trnK sequence [51]. Likewise, our data showed two groups of Chenapodium DHNs, one that was clustered with Atriplex in a superclade resolved with an 80% bootstrap value and another group clustered into two major clades with Beta and Spinacia.
Based on an extensive phylogenetic analysis using maximum likelihood and utilizing 59 protein-coding genes [52], a tight clustering was reported for both Spinacia and Chenapodium followed by Beta among 11 genera from the Chenopodiaceae family. Likewise, Spinacia and Chenapodium were found to cluster together based on the atpB-rbcL spacer sequence [48]. In addition, and based on the flowering locus (FT), orthologs were clustered together between Beta and Chenopodium [53]. These clusters agree with our phylogenetic analysis of DHNs.
The cloned DHN from A. halimus (MH591431) showed several SNPs compared with the published DHN genes from A. halimus (KF578414) and A. canescens (JN974246). In addition, the highly conserved S-segment available in DHN genes from A. halimus and A. canescens consists of six residues of the amino acid serine (SSSSSS) [16,17], while the S-segment in the DHN gene from A. halimus (AYH52686) was found to have C instead of S at the third position (SSCSSS). Moreover, the PCR amplified DHN gene from A. nummularia showed two bands using gel electrophoresis, which could be an indication of the presence of two paralogous dehydrins in this species. Multiple dehydrins have already been recorded for several plant species. This can start from two copies as in Vitis vinifera and can go up to fifteen copies as in Malus domestica [54].
The sequencing of both fragments from A. nummularia revealed novel forms in the S-segment. The first upper band in A. nummularia (AYH52689) with a size of 761 bp gave an S-segment with three cysteines and three serines (SCCCSS), while the second lower band in the same species A. nummularia (AYH52690) with a size of 495 bp gave an S-segment rich in serein residues with threonine residues at the second position (STSSSS). On the contrary, A. gardneri var. utahensis (AYH52691) showed another variant in the S-segment with isoleucine replacing a serine residue at the second position (SISSSS). Although the S-segment in the DHN is highly conserved, however our data revealed novel variants reported for the first time, which is consistent with earlier works showing other variants such as the glycine-containing S-segment (S2G) in EjDHN2 from Eriobotrya japonica [55], the S2N available in A. thaliana (CAA62449) or the S2G form present in DHN3 from Coffea canephora [56].
The SK-segment showed more variability between species because it extends for a longer stretch (40-42 residues) and due to the presence of a gap in the gene structure [48]. This can lead to changes in the position of the S-segment between 1-18, while the Ksegment runs between 27-42 positions. The most frequent SKn-segment present in the available DHNs is the YnSKn form (85%), while the SKn form is less frequent. Nonetheless, additional very rare forms were also recorded for plant dehydrins, e.g., the SKKS form present in Stellaria longipes [57] and the SKKYKY form present in Cerastium arcticum [58]. It is worth mentioning that the K-segment was found to have a protective role against both biotic and abiotic stresses, e.g., in grapes [59].

Conclusions
In this study, we found that most DHNs from the Azraq desert have a unique protein structure and presumably function. This could enable saltbush plants to survive the prevailing harsh conditions in the desert. On the other hand, the related DHNs from other species also showed unique and novel motifs (e.g., the S-segment) that would make the original plant be adapted to the specific conditions they live in. Therefore, the obtained data could guide future work to resolve holistic DHN interactomes with membranes, DNA, and other proteins to identify the uniqueness of each protein which would aid in mitigating different Atriplex spp. to different environmental stresses.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analysis, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.