High-affinity potassium transporters (HKTs) play a central role in maintaining Na⁺/K⁺ homeostasis in cereals under salinity stress. This study aimed to systematically characterize the barley HKT family (HvHKTs) and integrate evolutionary, regulatory (cis-elements and miRNAs), and expression evidence to identify key candidates associated with salt tolerance. A genome-wide identification and characterization of HvHKT genes was performed, including analyses of protein features (length, isoelectric point, and hydropathy), phylogeny, conserved motifs, and gene structures. Regulatory potential was assessed through promoter cis-element analysis, subcellular localization prediction, functional enrichment, protein–protein interaction (PPI) network analysis, and construction of a predicted miRNA–target network. In addition, salt stress-responsive expression patterns were examined across tissues, salinity levels, and time points, and qRT-PCR was used to validate key ion homeostasis-related genes, including HVP1, HvSOS1, and HvNHX1. A total of 39 HvHKT genes were identified, encoding proteins of approximately 300–540 amino acids with alkaline isoelectric points and hydrophobic GRAVY values, consistent with membrane-associated cation transporters. Phylogenetic analysis classified the HvHKT family into three clades corresponding to HKT1- and HKT2-type transporters, indicating lineage-specific expansion in barley. Conserved motif and gene structure analyses revealed a shared core architecture together with clade-specific variation, suggesting functional divergence. Promoter regions were enriched in stress-, hormone-, and light-responsive cis-elements, indicating multilayered transcriptional regulation. Subcellular localization predictions predominantly supported plasma membrane targeting, with some members also associated with vacuolar or other organellar compartments, while functional enrichment and PPI analyses linked HvHKTs to ion transport and homeostasis. The predicted miRNA–target network displayed a non-random hub-and-module organization, suggesting that a small set of hub miRNAs may exert broad post-transcriptional regulation, whereas additional miRNAs may provide more specialized or redundant control. Expression profiling under salinity stress revealed clear tissue-, dose-, and time-dependent regulation of HvHKT genes, with several members strongly induced in roots under high NaCl conditions. qRT-PCR further confirmed dose-dependent upregulation of HVP1, HvSOS1, and HvNHX1, supporting proton-driven vacuolar sequestration and plasma membrane Na⁺ efflux. Within the HvHKT family, HvHKT38, HvHKT36, HvHKT39, and HvHKT19 were the most prominent responders, HvHKT25, HvHKT27, and HvHKT30 showed moderate induction, and HvHKT31, HvHKT32, HvHKT33, and HvHKT34 displayed weaker or context-dependent responses. The stronger transcriptional activation observed under 400 mM NaCl suggested stress intensity-dependent tuning of the barley salinity response. The barley HvHKT family has undergone evolutionary expansion and functional diversification and is regulated through coordinated promoter architecture, miRNA-mediated control, and salt-responsive expression dynamics. Among these genes, HvHKT38, HvHKT36, HvHKT39, and HvHKT19, together with HVP1, HvSOS1, and HvNHX1, emerged as high-confidence candidates for functional validation and for breeding strategies aimed at improving salinity tolerance in barley. Graphic Abstract Schematic model of the multi-layer regulation of Hordeum vulgare high-affinity potassium transporter (HvHKT)-mediated ionic homeostasis under salinity stress. Promoter cis-regulatory elements, including ABRE (abscisic acid-responsive element), MYB/MBS (MYB-binding site), and MeJA (methyl jasmonate)-responsive motifs, enhance the transcriptional responsiveness of HvHKT genes. A small set of hub miRNAs (microRNAs), including miR6177, miR6190, miR6192, and the miR620x module, post-transcriptionally modulates HvHKT transcripts. Differential regulation between roots and shoots, as well as between early and late response phases, fine-tunes Na⁺ (sodium) retrieval, sequestration, and long-distance transport. Downstream, HvSOS1 (Hordeum vulgare Salt Overly Sensitive 1) mediates cytosolic Na⁺ efflux, whereas HvNHX1 (Hordeum vulgare Na⁺/H⁺ exchanger 1) and HVP1 (H⁺-translocating inorganic pyrophosphatase 1) promote vacuolar Na⁺ sequestration and energization, thereby restoring cellular ion balance under salinity stress

An Integrative Genome-Wide Study of Barley HKTs Links Gene Family Evolution to miRNA-Regulated Ionic Homeostasis Under Salinity

期刊论文
发表日期：

2026年5月4日

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Samar G. Thabet,
Fatmah Ahmed Safhi,
Ahmad M. Alqudah,
Amr Elkelish,
Nihal El Nahhas

摘要

High-affinity potassium transporters (HKTs) play a central role in maintaining Na⁺/K⁺ homeostasis in cereals under salinity stress. This study aimed to systematically characterize the barley HKT family (HvHKTs) and integrate evolutionary, regulatory (cis-elements and miRNAs), and expression evidence to identify key candidates associated with salt tolerance. A genome-wide identification and characterization of HvHKT genes was performed, including analyses of protein features (length, isoelectric point, and hydropathy), phylogeny, conserved motifs, and gene structures. Regulatory potential was assessed through promoter cis-element analysis, subcellular localization prediction, functional enrichment, protein–protein interaction (PPI) network analysis, and construction of a predicted miRNA–target network. In addition, salt stress-responsive expression patterns were examined across tissues, salinity levels, and time points, and qRT-PCR was used to validate key ion homeostasis-related genes, including HVP1, HvSOS1, and HvNHX1. A total of 39 HvHKT genes were identified, encoding proteins of approximately 300–540 amino acids with alkaline isoelectric points and hydrophobic GRAVY values, consistent with membrane-associated cation transporters. Phylogenetic analysis classified the HvHKT family into three clades corresponding to HKT1- and HKT2-type transporters, indicating lineage-specific expansion in barley. Conserved motif and gene structure analyses revealed a shared core architecture together with clade-specific variation, suggesting functional divergence. Promoter regions were enriched in stress-, hormone-, and light-responsive cis-elements, indicating multilayered transcriptional regulation. Subcellular localization predictions predominantly supported plasma membrane targeting, with some members also associated with vacuolar or other organellar compartments, while functional enrichment and PPI analyses linked HvHKTs to ion transport and homeostasis. The predicted miRNA–target network displayed a non-random hub-and-module organization, suggesting that a small set of hub miRNAs may exert broad post-transcriptional regulation, whereas additional miRNAs may provide more specialized or redundant control. Expression profiling under salinity stress revealed clear tissue-, dose-, and time-dependent regulation of HvHKT genes, with several members strongly induced in roots under high NaCl conditions. qRT-PCR further confirmed dose-dependent upregulation of HVP1, HvSOS1, and HvNHX1, supporting proton-driven vacuolar sequestration and plasma membrane Na⁺ efflux. Within the HvHKT family, HvHKT38, HvHKT36, HvHKT39, and HvHKT19 were the most prominent responders, HvHKT25, HvHKT27, and HvHKT30 showed moderate induction, and HvHKT31, HvHKT32, HvHKT33, and HvHKT34 displayed weaker or context-dependent responses. The stronger transcriptional activation observed under 400 mM NaCl suggested stress intensity-dependent tuning of the barley salinity response. The barley HvHKT family has undergone evolutionary expansion and functional diversification and is regulated through coordinated promoter architecture, miRNA-mediated control, and salt-responsive expression dynamics. Among these genes, HvHKT38, HvHKT36, HvHKT39, and HvHKT19, together with HVP1, HvSOS1, and HvNHX1, emerged as high-confidence candidates for functional validation and for breeding strategies aimed at improving salinity tolerance in barley.

Graphic Abstract

Schematic model of the multi-layer regulation of Hordeum vulgare high-affinity potassium transporter (HvHKT)-mediated ionic homeostasis under salinity stress. Promoter cis-regulatory elements, including ABRE (abscisic acid-responsive element), MYB/MBS (MYB-binding site), and MeJA (methyl jasmonate)-responsive motifs, enhance the transcriptional responsiveness of HvHKT genes. A small set of hub miRNAs (microRNAs), including miR6177, miR6190, miR6192, and the miR620x module, post-transcriptionally modulates HvHKT transcripts. Differential regulation between roots and shoots, as well as between early and late response phases, fine-tunes Na⁺ (sodium) retrieval, sequestration, and long-distance transport. Downstream, HvSOS1 (Hordeum vulgare Salt Overly Sensitive 1) mediates cytosolic Na⁺ efflux, whereas HvNHX1 (Hordeum vulgare Na⁺/H⁺ exchanger 1) and HVP1 (H⁺-translocating inorganic pyrophosphatase 1) promote vacuolar Na⁺ sequestration and energization, thereby restoring cellular ion balance under salinity stress