In silico approach to investigate the potential HKT gene responsive to salt stress in rice

Rice is frequently subjected to various environmental stresses, resulting in significant production losses, with drought and salinity are the leading causes of plant damage globally. This study aims to characterize and understand the func‑ tion of rice high‑affinity potassium transporters ( HKTs ) genes in response to salinity stress. Initially, the genome‑wide analysis was undertaken to reveal the evolutionarily conserved function of the OsHKT in higher plants. To investigate the transcription level of OsHKT during the vegetative and reproductive stages, two microarray datasets (GSE19024 and GSE3053) were analyzed, and salt‑treated samples were subsequently evaluated using real‑time PCR. Differentially expressed genes ( DEGs ) were identified from microarray datasets (GSE41650 and GSE14403), followed by construct‑ ing a DEG network that highlighted interaction partners of the OsHKTs . Genome mining of rice revealed 9 HKT genes, namely OsHKT1;1–1;5 and OsHKT2;1–2;4 . These genes exhibited a well‑conserved domain structure called TrkH . Com‑ prehensive phylogenetic and motif analyses clustered genes encoding HKT proteins into seven monophyletic groups, and the motifs were relatively conserved. Ka/Ks ratios indicated a high degree of purifying selection during evolution‑ ary time. Gene ontology findings suggested the involvement of OsHKT in stress response. Besides, several CRE motifs in the promoter regions of OsHKT have demonstrated their potential roles in abiotic stress responses. Furthermore, we analyzed the top 250 significant DEGs from the two datasets (p‑value < 0.05; fold two change ≥ 1 or ≤ − 1) to evalu‑ ate the relationship among the DEGs and HKTs . Three co‑expressed OsHKT genes were discovered to be upregulated in seedlings under salinity treatment, including OsP5CS2 , OsHAK1 , and OsNHX2 , whereas OsP5CS1 and OsHAK27 were downregulated. The transcripts of OsHKT were found to be differentially expressed in a tissue‑specific manner. Analysis of microarray datasets validated by real‑time PCR shows that OsHKT1;5 had a higher expression level, followed by OsHKT1;1 , OsHKT1;3 , and OsHKT2;1 after salinity treatment. In addition, several micro‑RNA targets in rice HKT genes regulate their expression in response to stress. This study paves the way for future investigation on genes and miRNA‑ target interaction in plants under environmental stresses, offering potential strategies to enhance stress tolerance in crops via targeted ion transport modification.


Introduction
Rice (Oryza sativa L.) is a staple food for nearly half the world's population, especially in tropical Latin America and most Asian countries (Shankar et al. 2016).Over the past decade, abiotic stresses have increased significantly due to environmental changes, land debasement, and declining water quality (Wassmann et al. 2009).Among these, soil salinity is one of the most devastating environmental stresses, causing significant reductions in cultivated land area, crop productivity, and quality (Shahbaz and Ashraf 2013).More than 800 million hectares of land are affected by salt, making up ~ 7% of the total land area (Munns et al. 2006).High salinity is estimated to affect 20% and 33% of total cultivated and irrigated agricultural lands worldwide.By 2050, it's projected that over 50% of agricultural land will be salinised due to low precipitation, high surface evaporation, weathering of native rocks, irrigation with saline water, and poor cultural practices (Jamil et al. 2011).Notably, most crops, especially rice, are salt-sensitive, prompting extensive research studies to uncover the mechanism of salt tolerance in crop species (Sytar et al. 2017).
Soil salinization hinders plant growth and development by increasing the concentration of sodium (Na + ) and chloride (Cl − ) ions in the soil, which disrupts seed germination, reproductive development, and vegetative growth (Munns and Tester 2008;Guo et al. 2015;Guo et al. 2018;Kamran et al. 2019).Under salt-affected areas, osmotic stress triggers physiological changes in plants, such as stomatal closure, increase in leaf temperature, inhibit photosynthesis (Awlia et al. 2016), and adverse effects on root architecture and cell wall properties (Geng et al. 2013;Julkowska et al. 2014;Feng et al. 2018).Excessive accumulation of Cl − ions can hinder photosynthesis, protein synthesis, and other essential enzyme activities (Yamaguchi et al. 2013;Hasanuzzaman et al. 2018), and ultimately affects premature leaf senescence and cell death in plants due to its toxicity (Munns et al. 2006;Munns and Tester 2008;Roy et al. 2014).Despite being toxic at high concentrations, Na + plays a role in osmoregulation and is a substitute for potassium (K + ) under low K + conditions due to their similar physiochemical properties.The use of Na + , notwithstanding, requires tight control over K + and Na + uptake, transport, and compartmentalization, which becomes crucial in states of high Na + concentration in plant vascular tissue (Flowers 1985;Hasegawa et al. 2000;Mühling and Läuchli 2002).Maintaining the K + /Na + ratio is fundamental for plant longevity, emphasizing the importance of Na + transport, water direct, and signaling atoms under salt stress exposure (Hilker and Schmülling 2019;Wang et al. 2019).
Maintaining the Na + /K + ratio in the cytosol for metabolic processes and salinity tolerance in plants is crucial because Na + can disrupt the K + balance in plants (Assaha et al. 2017).Several types of Na + transporters have been reported to play critical roles in Na + homeostasis during salinity.These, include the sodium-hydrogen antiporter (NHX) involved in vacuolar sequestration of Na + , salt overly sensitive (SOS) responsible for root avoidance of toxic concentration, the non-specific cation channel (NSCC) which provides the main pathway for Na + uptake and translocation into the root at high NaCl concentrations, and high-affinity potassium transporter (HKT) that aids in the removal of Na + from the cell (Demidchik and Maathuis 2007;Quan et al. 2018;Yang and Guo 2018;Arabbeigi et al. 2019;Bernstein 2019).In addition, the presence of K + -transporting membrane proteins, such as AKT/KAT-type channels, HKT-type transporters, and HAK/AT/KUP-like transporters has also been observed to participate in low and/or high affinity K + uptake systems of rice (Mäser et al. 2001;Golldack et al. 2002).
High-affinity potassium transporters (HKTs) are membrane proteins that play a vital role in facilitating cation transport across the plasma membranes of plant cells (Waters et al. 2013) and are also crucial in managing salt tolerance and mitigating the effects of salinity on plants.HKTs prevent the entry of Na + particles into shoot tissues by eliminating Na + from the xylem and regulating Na + and K + levels in parenchyma cells (James et al. 2006).The high-affinity K + -update system was first discovered in wheat HKT1 protein with some others encoded as Na + uniporters (Uozumi et al. 2000) and Na + and K + co-transporters (Schachtman and Schroeder 1994).HKT gene family has also been widely found in eudicotyledon and monocotyledon plant species, such as Arabidopsis (Maser et al. 2002), barley (Haro et al. 2005), eucalyptus (Liu et al. 2001), grapevine (Jabnoune et al. 2009), ice plant (Su et al. 2003), rice (Horie et al. 2001;Garciadeblás et al. 2003), and wheat (Schachtman and Schroeder 1994).HKT is divided into two subfamilies.HKT subfamily 1 is a Na + transporter, while HKT subfamily 2 is merely found in monocotyledon plants and is responsible for transporting K + and Na + across the cells.According to a phylogenetic study (Platten et al. 2006), both HKT subfamilies have a distinct conserved amino acid residue in the first pore loop of the amino acid sequence, with subfamily 1 members containing the amino acid Ser-Gly-Gly-Gly, while serine is replaced by glycine in subfamily 2 members, which designated as Gly-Gly-Gly-Gly (Maser et al. 2002;Garciadeblás et al. 2003).While the presence of glycine permits the transport of Na + and K + depending on the external ion concentration, the presence of serine favors the transport of Na + over other cations.
Despite the critical role of OsHKT as Na + and K + transporters under salinity stress, there is a need for more comprehensive studies.In this study, we investigated the functions and evolutionary relationship of OsHKT gene family members in rice with eudicot plants by performing in silico analysis on the publicly available sequenced genome.Our findings on gene structure, chromosomal localization and duplication pattern, promoter analysis, expression patterns, miRNA pattern, and co-expression networks suggest that OsHKT genes are involved in ion homeostasis through Na + and K + transport in response to the salinity.Our results offer a valuable resource for functional studies of the OsHKT gene to mitigate abiotic stress problems in rice.

Identification of OsHKT family members in rice
To obtain the potential candidate HKT amino acid sequences in rice, the Hidden Markov Model (HMM) profiles of the conserved HKT domain (PF02386) were downloaded from Pfam (http:// pfam.sanger.ac.uk/) (Mistry et al. 2021).HMM profiles are powerful probabilistic models designed to capture the evolutionary variations in a group of related sequences.The BLASTP search using the HMM profile was carried out to scan the protein database on the MSU Rice Genome Annotation Project (MSU-RGAP) (rice.plantbiology.msu.edu),Phytozome (https:// phyto zome.jgi.doe.gov) (Goodstein et al. 2012), Ensemble Genomes (https:// plants.ensem bl.org/ Oryza_ sativa/ Info/ Index) (Monaco et al. 2014) with the parameters of an E-value threshold of -1 and the BLOSUM62 comparison matrix.Also, HKT genes were retrieved from the National Center for Biotechnology Information (NCBI) (https:// www.ncbi.nlm.nih.gov) (Sayers et al. 2021) using the keyword search 'HKT' .The list of HKT genes was then integrated, followed by the removal of redundant genes.The Online CD-search tool of NCBI (https:// www.ncbi.nlm.nih.gov/ Struc ture/ cdd/ wrpsb.cgi), Pfam (http:// pfam.sanger.ac.uk), and Simple Modular Architecture Tool (SMART) (http:// smart.embl-heide lberg.de/ smart/ batch.pl) (Letunic and Bork 2018) was used to reconfirm the class of predicted HKT proteins.

Phylogenetic analysis
In this study, two phylogenetic trees were constructed among the HKT gene family members in (i) monocotyledons (rice, wheat, and maize) and (ii) eudicotyledons (Arabidopsis, grape, and tomato).The amino acid sequences of HKT proteins, namely OsHKT (rice), TaHKT (wheat), ZmHKT (maize), AtHKT (Arabidopsis), VvHKT (grape), and SlHKT (tomato) were downloaded from Phytozome v13, Ensemble Plants, and NCBI.Multiple sequence alignments for each analysis were performed using the ClustalX2.0(Li et al. 2015) tool with the default parameters.The phylogenetic tree analysis was then constructed using MEGA X software with the Maximum Likelihood (ML) method, and the bootstrap value was set to 1000 replicates with a complete deletion mode (Kumar et al. 2018).

Gene structure and conserved motifs analysis
We used the Gene Structure Display Server 2.0 (http:// gsds.gao-lab.org) to visualize the exon-intron arrangement of the OsHKT gene by aligning the genomic DNA with the corresponding cDNA sequences (Hu et al. 2015).To further support the evolutionary relationship, conserved motifs in the OsHKT protein sequences were identified using the MEME suite version 5.3.3(https:// meme-suite.org/ meme/) (Bailey et al. 2009).The parameters used for motif discovery were as follows: site distribution models = zoops, number of motifs = 10, width of motifs > 6 and < 50, and sites of motif > 2 and < 600.The function of each motif was then searched against the CDsearch tool of the NCBI database with a default E-value cutoff of 0.01 (Marchler-Bauer et al. 2017).MyHits (https:// myhits.sib.swiss/) was also used to annotate the motif sequence for functional prediction.

Chromosomal localization and gene duplication analysis
To illustrate the gene locations on the rice chromosome, the chromosomal positions of OsHKT genes were acquired from the phytozome and mapped using TBtools software (Chen et al. 2020a).Two or more genes located on the same chromosome represent the possibility of tandem duplication, whereas genes on different chromosomes indicate segmental duplication (Zhu et al. 2014;Nasim et al. 2016).Therefore, the tandem and segmental duplications of the OsHKT gene were observed based on their locations in the chromosome.To further calculate the evolutionary time of the OsHKT gene family, the non-synonymous (d N or Ka) and synonymous (d s or Ks) values were calculated using PAL2NAL (Suyama et al. 2006).The duplication time of the gene pairs was estimated using the formula of the synonymous mutation rate of substitution per synonymous site per year as follows: T = Ks/2x, (x = 6.56 × 10 -9 ), where, T = time of divergence, Ks is the synonymous substitution per synonymous site, and x is the mean rate of synonymous substitution (Yuan et al. 2015).The d s /d N ratio was used to detect the selective pressure on the HKT genes and by aligning the DNA-coding sequence of the HKT genes in rice to identify site-specific positive or purifying selection by the Selecton Server (Stern et al. 2007).

Subcellular localization, transcription factor binding sites (TFbs) and cis-regulatory elements (CREs) analysis, and gene regulation of OsHKT family in rice
To predict the subcellular localization of the OsHKT protein, the protein sequences were blasted against eukaryote protein sequences in the CELLO2GO webserver with an E-value of 0.001 (http:// cello.life.nctu.edu.tw/ cello 2go/) (Yu et al. 2014).The 1.5 kb upstream of the genomic sequences were retrieved from the Phytozome to identify the promoter regions of the OsHKT.Furthermore, the transcription binding sites were predicted using 1.5 kb genomic sequences as input data and searched against a multiple promoter analysis database, PlantPAN 2.0 (http:// plant pan2.itps.ncku.edu.tw/) (Chow et al. 2016) and CREs using PlantCARE.The PlantRegMap (http:// plant regmap.gao-lab.org) was utilized to retrieve gene regulation information containing interaction between transcription factors that regulate the OsHKT gene (Tian et al. 2019).The interactions between the TF and OsHKT genes were then visualized by using Cytoscape v3.8.2 (Shannon 2003).

Genome-wide expression analysis of the OsHKT family in rice
The expression datasets for the OsHKT gene family in 22 tissues for the indica rice variety Minghui 63 were extracted from the Affymetrix rice microarray data in the Collection of Rice Expression Profiles (CREP) database under accession number GSE19024 (Wang et al. 2010).For salinity treatment, we used the microarray dataset GSE3053 from NCBI GEO, which includes salt-tolerant FL478 and salt-sensitive IR29 genotypes (Walia et al. 2005).The strongest signal was used using multiple probe sets for a single gene.GEO expression datasets and the treatment log 2 fold change dataset were normalized using a gene-wise normalization combination technique.To cluster the expression data of OsHKT under salinity and tissues, we generated the heatmap using TBtools software (Chen et al. 2020a).

Array data collection acquisition and identification of DEGs
This study retrieved two sets of microarray series containing expression profiles from the GEO database (Clough and Barrett 2016).The keywords "salinity" and "rice" were selected to search GEO datasets for related gene expression profiles.GSE41650 consists of 27 samples, nine of which are control (7-day-old seedlings without treatment) and 18 are salinity (7-day-old seedlings with salinity treatment).GSE14403, on the other hand, contains 23 samples, including 11 untreated root and 12 salt-treated root samples as control and salinity, respectively (Cotsaftis et al. 2011).Both datasets were obtained using the platform GPL2025 [Rice] Affymetrix Rice Genome Array.
To examine the differentially expressed genes (DEGs), the online statistical tool GEO2R was utilized (Barrett et al. 2012).The GEO2R inbuilt methods, such as the T-test and Benjamini and Hochberg (false discovery rate), were applied to calculate the p-value and false discovery rate (FDR) determining the DEGs between control and salinity group (Aubert et al., 2004).The principal criteria of |log (fold change)|> 1 and p < 0.05 were applied to identify significant DEGs from the dataset.The DEGs were considered upregulated if the logFC ≥ 1 and downregulated if the logFC ≤ − 1.

Establishment of OsHKT protein networks and gene ontology (GO) annotation
A protein-protein interaction (PPI) network of differentially expressed HKTs was constructed using the Cytoscape String App (Doncheva et al. 2019).A confidence score ≥ 0.4 was employed to retrieve the PPI information of statistically significant DEGs from the STRING database.The PPIN of HKTs was then visualized using Cytoscape software v3.7.1 (Shannon 2003).To annotate the OsHKT genes, all the protein sequences were blasted against eukaryote protein sequences in the CELLO2GO webserver with an E-value of 0.001 (Yu et al. 2014).The results were then categorized into biological processes, molecular functions, and cellular components.

miRNA target site prediction of OsHKT proteins in rice
First, mature miRNA was obtained from the PmiREN website (https:// www.pmiren.com/) to identify the OsHKT gene family's target locations in rice (Guo et al. 2022).Next, we used the web server program PsRNA (https:// www.zhaol ab.org/ psRNA Target/) with the default settings to search the CDSs of the OsHKT genes against mature miRNAs (Dai and Zhao 2011).Cytoscape was used to build the networks connecting the anticipated miRNAs (Shannon 2003).

Plant materials and treatment
Mature seeds of pokkali and IR64 were used for expression analysis.Seeds were then sterilized with 5% sodium hypochlorite solution for 10 min and rinsed with distilled water 5-6 times.Next, sterile seeds were submerged in deionized water at 30 °C for two days before being placed in a growth chamber and incubated for 24 h at 28 °C.The seedlings were grown in hydroponic solution for 21 days according to IRRI protocol (Yoshida et al. 1976).The uniform 21-day-old seedlings were imposed to 100, 150, and 200 mM NaCl with control.Tissues were collected immediately for control and after 24h NaCl treatments for RNA isolation.

RNA isolation, cDNA synthesis and qRT-PCR
According to the manufacturer's instructions, total RNA was extracted from each genotype using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, USA).Total RNA was tested for purity and integrity with a Nanodrop.The RNA sample was taken at an A260/280 ratio of 1.8-2.0 and an A260/230 ratio of 2.0-2.2, and it has been kept at −80 °C until further usage.Following the manufacturer's instructions, the first strand of cDNA was synthesized with a HiScriptIII First Strand cDNA Synthesis Kit (Vazyme Biotech, China).For two minutes at 42 °C, 100 ng of total RNA was combined with 2 µL of 5 × gDNA Mix wiper and RNase-free sterile water.The cDNA synthesis mixture contains 4 µL of RNAse-free sterile water, 1 µL of Oligo (dT) 20 VN, 1 µL of Random hexamers, and 2 µL of 10 × RT Mix.The mixture was incubated at 37 °C (15 min) and 85 °C for 5 s.The resultant cDNA products are frozen at −80 °C until needed.THUNDERBIRD ® SYBR ® qPCR mix (TOYOBO, Japan) was used to perform qPCR amplification on cDNA aliquots of 3 µL in 20 µL reaction volumes with gene-specific primer and actin as an internal control (Supplementary Table 1) in 96-well plate Applied Biosystems 7500 Fast Real-Time PCR system.The 2 −∆∆CT method was used to analyze the relative expression of genes (Livak and Schmittgen 2001).

Statistical analysis
The statistical analysis was conducted utilising analysis of variance (ANOVA), and the means were compared using the Least Significant Difference (LSD) at a significance level of P ≤ 0.05, employing the R program.The steps taken to analyze the HKT family members in rice are depicted in Fig. 1.

Genome-wide identification of HKT family proteins in rice
In our study, nine HKT genes were identified in rice, namely OsHKT1;1, OsHKT1;2, OsHKT1;3, OsHKT1;4,  OsHKT1;5, OsHKT2;1, OsHKT2;2, OsHKT2;3, and  OsHKT2;4.Among them, OsHKT1;2 and OsHKT2;2 are known as pseudogenes in Oryza sativa Nipponbare (Horie et al. 2001).The HKT family members in rice consist of a highly conserved domain structure called TrkH, a cation transport protein domain responsible for actively transporting sodium ions into the cell.Figure 2 illustrates the presence of the TrkH conserved protein motif within the HKT family in rice.The OsHKT family genes exhibit significant variations in the size and properties of the encoded proteins (Table 1).We predicted OsHKT1;5 to be the longest HKT protein, with 554 aa, whereas OsHKT1;4 has the shortest length of amino acids, with 500 aa.A wide range of predicted molecular weights was found among OsHKT genes, ranging from 54.24 kDa to 60.22 kDa, and an isoelectric point (pI) ranging from 8.74 to 9.49.Minor differences in molecular weight and theoretical isoelectric point are observed among HKT proteins, suggesting subtle differences in physical and chemical characteristics in rice.The grand average of hydropathicity (GRAVY) values of positive and negative residues indicates a protein's hydrophobicity and hydrophilicity, respectively.All the HKT proteins in rice showed positive GRAVY values, indicating that OsHKT proteins were hydrophobic.OsHKT genes, namely OsHKT1;1, OsHKT1;3, OsHKT2;1, and OsHKT2;2 was predicted to be stable proteins based on the cut-off instability index < 40, while the OsHKT1;4, OsHKT1;5, OsHKT2;3 and OsHKT2;4 was unstable with an instability index > 40 as shown in Table 1.Protein sequence similarity indicated that OsHKT2;1 and Po_OsHKT2;2 showed the highest levels of protein sequence similarity (82.58%), whereas OsHKT1;1 and OsHKT1;4 showed the least protein sequence similarity (31.84%) (Supplementary Table 2).In addition, OsHKT family proteins were found to be in the plasma membrane and comprise an equal number of transmembrane helices, as shown in Table 1.

Phylogenetic analysis and identification of conserved motifs and gene structure of OsHKT family proteins
A phylogenetic tree was constructed to gain insights into the evolutionary relationship among the OsHKT genes (Fig. 3A).The constructed phylogenetic tree is composed of two major monophyletic branches, comprising four paralogous pairs of OsHKT such as OsHKT1;1-OsHKT1;2, OsHKT1;4-OsHKT1;5, OsHKT2;1-Po_OsHKT2;2 and OsHKT2;3-OsHKT2;4.This reflects the highly conserved nature of the OsHKT family gene, particularly among the OsHKT1 and OsHKT2 groups.Also, MEME analysis discovered ten distinct motifs in the OsHKT protein sequences, with motif lengths ranging from 24 to 50 amino acids (Fig. 3B).Nine out of ten motifs appeared in all the OsHKT proteins, suggesting that both OsHKT1 and OsHKT2 are relatively conserved; however, one motif was discovered to be uniquely present in all genes within the monophyletic group of OsHKT2, indicating OsHKT2 to have a distinct function compared to OsHKT1.
To infer the function of each motif, we further annotated the motifs using motif scan and CD-search tools.Motifs 1-7 and 9 were mainly annotated as TrkH (cation transport protein).Several site-specific motifs were also detected among the motifs.For instance, Motif 1 is associated with the N-glycosylation site and the protein kinase C phosphorylation site.Motif 4, on the other hand, is related to the N-myristoylation site, and Motif 5 is associated with the cAMP-and cGMP-dependent protein kinase phosphorylation sites.Motif 7 is related to  the tyrosine kinase phosphorylation site, and Motif 8 is associated with the casein kinase II phosphorylation site.Motif 9 is also annotated for the protein kinase C phosphorylation site, and motif 10 is related to the cAMPand cGMP-dependent protein kinase phosphorylation sites and the casein kinase II phosphorylation sites.The absence of motif 10 in OsHKT1 family genes could be attributed to the diversification and loss of specific sequences during the evolution of rice.This phenomenon might have resulted in distinct functions between the OsHKT1 and OsHKT2 groups.
The diversification and arrangement of gene structures have had a significant impact on the evolution of gene families.Figure 4 depicts detailed information on introns, exons, and untranslated regions of OsHKT genes.Exons are the coding regions that code for amino acids and are separated by noncoding regions called introns.Introns play essential roles in various cellular processes, including genomic recombination, which can lead to gene rearrangements and contribute to the evolution of genes and species.The monophyletic group of OsHKT1 genes is composed of 2-3 exons and separated by 1-2 introns.The paralogous pair of OsHKT1;4-OsHKT1;5 have the same number of exons and introns, while OsHKT1;1 and OsHKT1;3 have a variable number of introns, with 1 and 2 introns, respectively.Two introns were identified for the OsHKT2 genes.The variable numbers of introns in OsHKT members indicated the possibility of loss and gain of exons during evolution.This may explain the functional variations among members despite being grouped in a similar phylogenetic clade.

Chromosomal localization, gene duplication and detection of selection
In this study, HKT genes were mapped on rice chromosomes.Specifically, two genes (OsHKT1;5 and OsHKT2;3) were found to be located on chromosome 1, followed by one gene (OsHKT1;3) on chromosome 2, two genes (OsHKT1;1 and OsHKT1;4) on chromosome 4, and two genes (OsHKT2;1and OsHKT2;4) on chromosome 6, as demonstrated in Fig. 5.The study provides valuable information about the genomic distribution of the HKT genes.For example, two or more OsHKT genes on the same chromosome may occur due to tandem duplication events, while genes on different chromosomes suggest the possibility of segmental duplication (Nasim et al. 2016;Zhu et al. 2014).To further understand the evolutionary mechanism of OsHKT genes, we found that two gene pairs (OsHKT1;4/OsHKT1;5 and OsHKT2;3/ OsHKT2;4) were the results of segmental duplications, implying the possible expansion events of the HKT gene family in rice (Fig. 5).The selection pressure on rice HKT genes during their evolutionary process was evaluated to support this hypothesis.The non-synonymous (d N or Ka) and synonymous (d s or Ks) substitution rates, as well as the Ka/Ks ratio and the approximate date of duplication using the Ks values, were calculated (Table 2).The Ks value of two pairs of segmented duplicates (OsHKT1;4/ OsHKT1;5 and OsHKT2;3/OsHKT2;4) ranges from 0.0688 Mya to 27.0803 Mya.Meanwhile, we discovered that the duplication times for segmental duplicates range from 5.2439 Mya to 2064.0473Mya.The Ka/Ks values for segmental duplication were less than 1 (0.0168 to 0.5131), indicating OsHKT genes have been subjected to intense purifying selective pressure.

Comparative analysis of rice HKT family genes with Wheat, Maize, Arabidopsis, Tomato and Grape
To see how the HKT family genes in rice and other monocots and eudicots have changed over time, a maximum likelihood phylogenetic tree was made from fulllength sequences of amino acids (Fig. 6).Based on our phylogenetic analysis, nine OsHKTs, seven TaHKTs, three ZmHKTs, one AtHKTs, two SlHKTs, and six VvHKTs were clustered into seven monophyletic groups I-VII (Fig. 6).Our evolutionary study also supports the 7-classification of the HKT gene family in rice and other organisms based on the conservation of their TrKH domain structure.The OsHKT genes were discovered to be clustered with other plant HKT genes, except for groups III and VII.Two members of the rice (OsHKT1;1, and OsHKT1;2) proteins belonged to group I. OsHKT1;4 clustered with one wheat (TaCS2A02G430600) and one maize (Zm00008a006337), and OsHKT1;3 clustered with two wheat (TaC-S6D02G144500 and TaCS7B02G182600) HKT proteins in group II and IV, respectively.Group V consists of one rice (OsHKT1;5) one maize (Zm0008a011700) and two wheat (TaCS4B02G370800 and TaCS7D02G361300) HKT proteins.Two OsHKTs (OsHKT2;3 and OsHKT2;4)  were found to be clustered in a similar monophyletic group VI, together with TaHKT (TaCS7D02G411300) and ZmHKT (Zm0008a020484).On the other hand, group VI also comprised two rice (OsHKT2;1 and Po_ OsHKT2;2) and one wheat HKT (TaCS7D02G411200) protein.Group VI has the most significant number of HKT genes.From phylogenetic analysis, OsHKT genes were highly conserved among monocots and dicots, especially with TaHKT and ZmHKT proteins.Several paralogous genes were also clustered in the same monophyletic group, such as OsHKT1;1/OsHKT1;2 in group I, OsHKT2;1/Po_OsHKT2;2 and OsHKT2;3/ OsHKT2;4 in group VI, two sets of wheat TaHKT in group V (TaCS4B02G370800/TaCS7D02G361300), IV (TaCS6D02G144500/TaCS7B02G182600) and one set of grape VvHKT (VvGSVIVT01010921001/ VvGS-VIVT01010922001) in group III, indicating species-specific duplication events of HKT genes.The orthologous gene pair OsHKT1;5/Zm00008a011700 was also identified between rice and maize in group V with 68.13% sequence similarity (Table 2).Further, the divergence of the orthologs between rice and maize HKT genes was investigated by calculating the Ka/Ks ratio.The result indicated that the Ka/Ks ratio of the orthologous gene pair was less than 1 (0.11), revealing purifying selection.The orthologous gene OsHKT1;5/Zm00008a011700 exhibits a conserved gene organization, as it shares the same number of introns.Additionally, the orthologous Fig. 6 Phylogenetic trees of full-length HKT proteins in rice, wheat, maize, Arabidopsis, tomato, and grape.Group V indicates an orthologous pair of rice HKT genes gene also showed a Ks value of less than 2.0, indicating a higher association with segmental duplication (Table 2).
Overall, this study revealed that the early rice HKT gene duplication event was observed in maize as compared to other plant species.

Analysis of putative TFbs of rice HKT family
The cis-acting regulatory elements (CREs) play a significant role in regulating the expression of genes in response to stress, light, and growth.To understand the interaction between transcription factors and binding sites of OsHKT genes, we predicted 1.5 kb upstream regions using plant promoter databases, PlantPAN 2.0 and Plant-CARE.Further, PlantRegMap was used to retrieve transcription factor information that regulates the OsHKT genes.In our study, we discovered nine important binding sites, including WRKY, bHLH, bZIP, MYB, AP2/ERF, GATA, B3, Dof, and C2H2 that were highly distributed in all the promoter regions of OsHKT genes (Fig. 7).The AP2/ERFbs responsive elements were highly abundant in the OsHKT gene promoters, followed by B3, GATA, and bZIP.The highest number of binding sites was found in OsHKT1;5, while the lowest was found in OsHKT2;4 (Fig. 8A).To better understand the regulatory mechanisms of OsHKT genes, the CREs were predicted using the PlantCare databases.A large number of CREs were found in the promoter region of OsHKT genes that are known as light-responsive elements such as GT1-motif, as-1, G-box, and TCCC-motif; hormone-responsive elements (CGTCA/TGACG-motif, ERE, ABRE, TCA and GARE); environmental stress-responsive elements (ARE, LTR, MBS, TC-rich repeats, W-box, DRE, STRE, MYB, and MYC) and plant growth and development-related elements (HD-Zip, AT-rich, CAT box, O2 site, and AAGAA-motif ).Detailed information on the CREs is presented in Figs.8B and 9.

Expression pattern of HKT genes in rice
To better understand OsHKT genes' response across the whole rice life cycle, we analyzed the expression patterns  3).The expression levels of OsHKT genes could be divided into two groups (Fig. 11A).Group I consists of two genes (OsHKT1;1 and OsHKT1;3) that have shown higher transcript accumulations, whereas OsHKT1;1 has the highest expression level in the entire rice life cycle and OsHKT1;3 has a high expression level in seedlings, shoots, leaves, sheaths and stamen.On the other hand, five genes belonged to group II, namely OsHKT1;5, OsHKT2;1, OsHKT2;4, OsHKT1;4 and OsHKT2;3.OsHKT1;5 has shown high expression levels in both vegetative and reproductive stages, such as seedlings, roots, stems, panicles, and spikelets.Other genes from group II exhibited low expression signals.
For the salinity treatment, the microarray data were analyzed to examine the responsiveness of OsHKT genes to salt stress.Two well-characterized salt-tolerant FL478 and salt-sensitive IR29 genotypes were used, with untreated seedlings serving as a control during the vegetative stage.Two major categories can be distinguished between the levels of OsHKT gene expression (Fig. 11B).Supplementary Table 4 displays the relative fold-change in OsHKT gene expression in response to salt treatment.Three OsHKT genes from group I (OsHKT1;5, OsHKT1;1, and OsHKT1;3) exhibited increased expression in the salt-tolerant FL478 and salt-sensitive IR29 genotypes under salinity stress.In group II, OsHKT2;1 displayed higher expression in both FL478 and IR29 genotypes, while the remaining genes (OsHKT1;4, OsHKT2;3, and OsHKT2;4) showed moderate expression in FL478 and lower expression in IR29 genotypes.
Real-time PCR was used to obtain further verification of the OsHKT gene expression pattern under salt stress (Fig. 12).Seven HKT genes were found to be strongly expressed in the salt-tolerant cv.Pokkali's roots and shoots, except OsHKT2;4.However, shoot OsHKT1;1, OsHKT1;3, OsHKT2;1, and OsHKT2;4 greater expression was found in pokkali.Meanwhile, the most substantial upregulation of OsHKT1;4, OsHKT1;5, and OsHKT2;3 was found in pokkali root after 24 h of 100 mM, 150 mM, and 200 mM salt treatment, respectively.On the other hand, salt-sensitive IR64 plant exhibited a significant decrease in the expression of all OsHKT family genes in both the root and shoot regions, except for OsHKT2;4.Interestingly, OsHKT2;4 displayed an increase in expression specifically in the roots region after 24 h.Remarkably, our findings strongly suggested OsHKT1;5, OsHKT1;1, OsHKT1;3, OsHKT2;1, and OsHKT2;3 as critical genes responsible for rice salinity tolerance.

Identification of DEGs and protein network of OsHKT
The GEO2R tool was used to find differentially expressed genes (DEGs) in salt stress from two gene expression datasets, GSE14403 and GSE41650.The DEGs with |log2FC|> 1 and |log2FC|< -1 and p < 0.05 were considered statistically significant, as demonstrated in Fig. 13A, B. From the DEG-based PPI network, a total of nine interactions from the GSE14403 and eight interactions from the GSE41650 datasets were discovered as demonstrated, in Fig. 13C, D, respectively.Among them, we found significant upregulation of OsHKT1 (also known as OsHKT2;1) to interact with pyrroline-5-carboxylate synthetase 2 (P5CS2), rice potassium transporter 1 (OsHAK1) and rice Na + /H + antiporters (OsNHX2).On the other hand, a downregulated OsHKT4, also called OsHKT1;1, significantly interacted with other downregulated DEGs, such as rice pyrroline-5-carboxylate synthetase 2 (OsP5CS2), pyrroline-5-carboxylate synthetase 1 (OsP5CS1), potassium transporter 27 (OsHAK27), and peroxidase 90 (prx90).This finding suggests that OsHKT1 and OsHKT4 play a significant role in regulating the concurrent expression of several genes under salt stress.

Functional GO annotation
The GO annotation analysis was conducted to describe the participation of OsHKT genes in the biological process and other functional relevance (Fig. 14).The GO annotation analysis demonstrated that the OsHKT genes were involved in transmembrane transporter activity at their molecular levels, and most of the OsHKT genes were found to be in the plasma membrane and nucleus, indicating their importance in cellular functioning activities.The OsHKT genes also play a crucial role in various biological functions, including response to stress, ion transport, and homeostatic processes.

Discussions
Salinity and drought are two of the most common abiotic stresses that plants frequently encounter and cause a negative impact on their growth, development, and production due to ion toxicity and physiological drought (Munns and Tester 2008;Tang et al. 2016).High-affinity potassium transporter (HKT) family proteins are anticipated to play an essential role in plant salt stress tolerance.HKTs were first identified as high-affinity potassium (K + ) transporters and were proven to transport sodium (Na + )  (Horie et al. 2009).HKTs are also responsible for Na + and K + transport as well as Na + -K + homeostasis in plants during plant development, making them potential goals for the development of salt tolerance in crops.Most of the research on the functional study of HKT family genes has focused on yeast and model plants such as Arabidopsis, as well as crops like wheat, maize, sorghum, barley, and eucalyptus (Liu et al. 2001;Rus et al. 2001;Haro et al. 2005;Munns et al. 2012;Ren et al. 2015;Li et al. 2019).However, a more thorough investigation needs to be into HKT family genes in rice.
In this study, we identified nine HKT genes, whereas eight are functional depending on the japonica and indica cultivars from the rice genome database.Based on the conserved domain search, we confirmed that TrkH is conserved in all the HKT genes (Fig. 2).The TrkH domain is a hydrophobic membrane protein vital in controlling Na + and K + movement in higher plants, contributing to enhanced salinity tolerance (Horie et al. 2009;Su et al. 2015).All OsHKT proteins have similar physiological properties, comprising equal transmembrane helices, and are mainly localized in the plasma membrane (Table 1).These findings indicate that HKT proteins have a close evolutionary connection with plants during biological evolution.The paralogous pairs of OsHKT genes found in subfamily 1 and 2 of the monophyletic tree also indicate that each subfamily's HKT genes undergo the same evolutionary process and serve the same functions, and potentially retain plant resistance to salt stress (Maser et al. 2002;Li et al. 2019).Furthermore, ten distinct motifs were discovered, and most of the motifs appeared in rice HKT proteins (Fig. 3B), suggesting that they are relatively conserved and have a strong evolutionary relationship (Singh et al. 2002).HKT genes have three exons and two introns (Fig. 4), demonstrating even more clearly that HKT genes in plants have been evolutionary conserved because the exon-intron arrangement has been utilized as supporting proof for developmental relationships between genes (Koralewski and Krutovsky 2011).In addition, gene duplication events are one of the critical factors that could provide a profound explanation for gene family expansion in plants (Moore and Purugganan 2005).The chromosomal location offers valuable information about tandem and segmental duplications of a specific family gene.Two or more genes located on the same chromosome reveal the possibility of tandem duplication, while genes situated on different chromosomes indicate segmental duplication events (Zhu et al. 2014;Nasim et al. 2016).Notably, two pairs of paralogous OsHKT genes (OsHKT1;4/OsHKT1;5 and OsHKT2;3/ OsHKT2;4) are segmentally duplicated, including OsHKT2;1/Po_OsHKT2;2 was discovered to be tandemly duplicated due to gene distributions on the same chromosome (Fig. 5).To understand functional sites and Fig. 15 The regulatory network between putative miRNAs and OsHKT genes functional protein alterations, selective pressure investigations are generally required as they reveal selective benefits for changing amino acid sequences in the protein (Morgan et al. 2010).The Ka/Ks value < 1 indicates the purifying selection, while the Ka/Ks ratio > 1 proposes the probability of a positive selection (Yang and Bielawski 2000;Bowers et al. 2003).Based on the Ka/Ks values for both segmental and tandem, they indicated that rice HKT genes have undergone intense purifying selection pressure (Table 2).The phylogenetic tree (Fig. 6) indicated that rice HKT proteins were related to monocot and dicot; however, they were more closely related to wheat and maize HKT proteins.The highly conserved cluster of HKT genes provided evidence that they perform similar functions rather than being the result of a series of evolutionary events (Zhang et al. 2013).One pair of orthologous OsHKT genes with maize (OsHKT1;5/ Zm00008a011700) revealed the purifying selection and showed the same intron numbers, which means conserved gene organization (Table 2).These findings suggest that the orthologous pair arose from the common inherited genes that existed before the divergence of the monocots and dicots.It is also mentioned that purifying selection played a vital role in the evolution of the HKT genes in other crop species (Zhang et al. 2019).
Transcription factors are pivotal in regulating the plant's response to abiotic stress by modulating the gene expression (Lindemose et al. 2013).Regulatory elements are crucial for detecting gene expression patterns, as regulatory elements control the expression of many genes through distinct binding sites (Mariño-Ramírez et al. 2009).WRKY, bHLH, bZIP, MYB, AP2/ERF, GATA, B3, Dof, and C2H2 type important binding sites that were highly distributed in all the promoter regions of OsHKT genes.TFs like WRKY, NAC, bHLH, bZIP, MYB, and AP2/ERF play vital roles in the responses to abiotic and biotic stress in many plant species (Lindemose et al. 2013;Das et al. 2019).Moreover, B3, a plant-specific transcription factor, has a variety of roles in the growth and development of plants (Peng and Weselake 2013), GATA is involved in light responsiveness (Behringer and Schwechheimer 2015), C2H2 type transcription factor plays a diverse role in plant growth and development as well response to stress (Yin et al. 2020) and Dof transcription factor also participates in many plant development stages and the response to different environmental stressors (Khan et al. 2021).The OsHKT genes possess a high abundance of stress-responsive cis-regulatory elements (CREs) such as MYB, MYC, MBS, W-box, ARE, STRE and DRE core.The expression of MBS, which is a binding site for MYB TF, changes in many plants when exposed to salt, suggesting that these plants are responding to salt stress (Hua et al. 2006).Moreover, members of this TF also influence the abscisic acid (ABA), polyethylene glycol (PEG), and SA-signalling pathways, which confer resistance to abiotic stresses (Ambawat et al. 2013).TGACG, CGTCA-motif, ERE, and ABRE were also abundant in the rice HKT promoter region.The next most common type of cis-acting elements were those that regulated growth and development, hormones, and light.The GT1-motif, as1, G-box, TCCC, and Box-4 are more frequently present in rice HKT promoters that were responsive to light.On the other hand, TGACG and CGTCA-motif were responsive to methyl jasmonate, ERE was responsive to ethylene, ABRE was responsive to abscisic acid (ABA), TCA was responsive to salicylic acid, TGA was responsive to auxins and P-box and GAREmotif were responsive to gibberellin.Plant hormones and other signalling pathways are important for adequate and integrated stress responses (Ryu and Cho 2015).ABA, methyl jasmonate, and ethylene have been suggested as factors governing adaptive responses to abiotic stimuli, while auxin, salicylic acid, and gibberellin are essential in growth and development.The presence of TGACG and CGTCA motifs suggests that methyl jasmonate may be involved in the regulation of the OsHKT gene.In line with this, a significant concentration of methyl jasmonate has been found in salt-tolerant rice cultivars (Kang et al. 2005).ERE-containing genes are regulated in the context of ethylene.It has been proposed that ethylene controls salt-responsive gene expression under salt stress (Verma et al. 2016).Most abiotic stress-responsive gene promoter regions comprise two cis-regulating elements, namely ABA-responsive elements (ABRE) and dehydration-responsive elements (DRE), both of which contain the core sequences ACG TGG /TC and TAC CGA CAT or A/GCC GAC , respectively (Kobayashi et al. 2004;Yamaguchi-Shinozaki and Shinozaki 2006) and the ABRE motif participates in the ABA-dependent gene expression under high-stress situations (Finkelstein 2013).According to previous research, there are interactions between ABA and methyl jasmonate at the MYC2 transcription factor, which is implicated in the control of gene expression under salt stress (Moons et al. 1997).Thus, cis-elements pertaining to ABA, ethylene, and methyl jasmonate suggest that these hormones have roles in OsHKTs in response to abiotic stresses.Various elements, such as W-box, WUN-motif, LTR, and TC-rich repeat also present in OsHKT promoter regions, are implicated in the salt stress response and defence (Gao et al. 2010;Li et al. 2013;Manimaran et al. 2017).According to promoter analysis, OsHKTs may respond to environmental stress and stimuli to regulate plant growth and development.
On the other hand, we have discovered that a wide range of transcription factors control OsHKT gene expression.These include C2H2 zinc finger protein, Myb transcription factor, Dof zinc finger domain-containing protein, DNA binding domain-containing protein, B3 domain-containing RAV and trihelix family-type protein, ARF, and dehydration-responsive transcription factors.Recent findings have reported that the expression of OsHKT1;1 has been positively regulated by OsMYBc.It binds to specific conserved DNA regions in the OsHKT1;1 promoter, modulating Na + concentration and preventing sodium toxicity in leaf blades (Wang et al. 2015).Knocking out OsMYBc also led to a decrease in the salt-induced expression of OsHKT1;1, and modifications in specific promoter regions resulted in reduced OsHKT1;1 promoter activity.To increase the expression of OsHKT1;5, the stable complex of OsSUVH7, OsMYB106, and OsBAG4 binds to the promoter of OsHKT1;5 (Wang et al. 2020).It has been shown that the bHLH transcription factor OsbHLH035 controls the expression of the genes OsHKT1;3 and OsHKT1;5.These findings indicate that OsbHLH035 positively influences the expression of OsHKT1;3 and OsHKT1;5 (Chen et al. 2018).Chen and his team identified a popular TF, PalERF109, as a positive regulator of the PalHKT1 gene expression (Chen et al. 2020b).In Arabidopsis, several TFs, such as AtbZIP24, ARR1, ARR12, and ABI4, have been found to regulate AtHKT1;1 expression (Yang et al. 2009;Mason et al. 2010;Shkolnik-Inbar et al. 2013).The observation indicated that AtbZIP24, ARR1, and ARR12 are negative regulators of AtHKT1;1 gene expression.Additionally, ABI4 TF also negatively regulates AtHKT1;1 gene expression, and the involvement of the abscisic acid signal transduction pathway in salt responses in Arabidopsis suggests that OsHKT1;5 may play a similar function in rice.The GT factors are a family of transcription factors found exclusively in plants and share a common DNA-binding trihelix domain.GT elements have A/T-rich core sequences and are highly degenerate ciselements.OsGTγ-1, OsGTγ-2, and OsGTγ-3 genes were upregulated in response to high salinity and other abiotic stimuli, suggesting a role for this subfamily transcriptional regulation of stress responses.At the vegetative stage, transgenic rice with an overexpression of OsGTγ-1 demonstrated an improvement in their salt tolerance (Fang et al. 2010).On the other hand, DREB TFs, which mainly bind with C-repeat/DRE (A/GCC GAC ), influence the expression of several cold or drought-inducible genes in an ABA-independent route, enhancing plant abiotic stress tolerance (Chen et al. 2008).Thus, these TFs have roles in OsHKTs in response to abiotic stresses.
In our study, OsHKT1;1 and OsHKT1;5 had higher expression levels in both vegetative and reproductive tissues.On the other hand, OsHKT1;3, OsHKT2;1, and OsHKT2;4 had higher expression in the vegetative stage, while OsHKT1;4 had lower expression in the vegetative stage (Fig. 11A).Also, both salt-tolerant and salt-sensitive genotypes have higher expression of OsHKT1;5, OsHKT1;1, OsHKT1;3, and OsHKT2;1 (Fig. 11B).Our real-time PCR results have confirmed that OsHKT1;5, OsHKT1;1, OsHKT1;3, OsHKT2;1, and OsHKT2;3 are crucial genes responsible for rice salinity tolerance (Fig. 12).The previous study on OsHKT1;4 also reported lower expression in a vegetative stage during stress (Suzuki et al. 2016).A rice QTL, SKC1, corresponded to OsHKT1;5 and maintained K + ion homeostasis under salt stress (Ren et al. 2005), and OsHKT1;5 mutants also showed Na + exclusion and protected leaf blades under salt stress (Kobayashi et al. 2017).OsHKT1;1 and OsHKT1;4 contribute to Na + exclusion from leaf blades under salt stress (Cotsaftis et al. 2012;Wang et al. 2015;Suzuki et al. 2016).OsHKT2;1/2 is also involved in Na + and K + co-transport under high salt concentrations and has been reported to maintain an appropriate ionic balance in Nona bokra (Oomen et al. 2012).The previous report also suggested that HKT genes play a vital role in response to salt stress in many plant species like wheat (Munns et al. 2012;Schachtman and Schroeder 1994), Arabidopsis (Sunarpi et al. 2005), maize (Ren et al. 2015) and barley (Mian et al. 2011).Thus, HKT genes in rice may be excellent candidate genes for accelerating transgenic research for salinity stress management in plant growth and development.
Proteins rarely function alone.A protein's activity can be activated, inhibited, or otherwise regulated through its interactions with other proteins or biological components.So far, no study has been disclosed identifying interaction partners for any HKT protein.The proteinprotein interaction exhibited exciting facts about the substantial contribution of OsHKT to numerous physiological functions (Fig. 13).Our findings show that rice HKT genes (OsHKT1;1 and OsHKT2;1) interact with the P5CS, which participates in salt stress tolerance and plays a vital role in proline biosynthesis (Zhang et al. 2014;Funck et al. 2020).Likewise, the OsHKT gene interacted with NHX1 and NHX2, a sodium/hydrogen exchanger-related protein that plays a central role during plant exposure to K + deficiency and high salinity (Fukuda et al. 2011;Barragán et al. 2012;Teng et al. 2017); HAK1, HAK23, and HAK27 are related to high-affinity potassium transporters that also transport rubidium.Furthermore, Os01g0893400 is a putative BTB and TAZ domain protein, and TPKC is an inward-rectifying potassium channel family protein that is known to be important in plant growth and development (Bhattacharjee et al. 2016;Wang et al. 2018).Interestingly, a small heat shock family protein, Hsp20/alpha-crystallin family protein, also interacts with rice HKT genes.Small HSPs are hypothesized to act as chaperones, protecting their targets against denaturation and aggregation when organisms are exposed to diverse biotic and abiotic stimuli.A recent study indicated that OsHSP20 exhibits molecular chaperone functions in vitro, and overexpression has been shown to improve heat and salt stress tolerance in E. coli, P. pastoris, and transgenic rice plants.It was also found that the N-terminal part of OsHSP20 is tightly linked to both in vivo stress tolerance and in vitro chaperone activity (Guo et al. 2020).Our results highlighted the significance of HKT transporters in rice salinity tolerance via their interactions with other proteins.
Plant cellular responses to abiotic stresses such as salinity, cold, and dehydration were revealed to be regulated by microRNA.Several miRNAs target genes that are actively involved in gene regulation or their associated transcription factors in response to stress.MiRNAs may play an essential role in reactions triggered by stress (Cheng and Long 2007;Sunkar et al. 2008).Fifteen Osa-miR11339 (bona fide mRNAs from rice) found in rice HKT genes represent lipid metabolisms in rice by targeting the terpene synthase gene (LOC_Os07g11790) (Baldrich et al. 2015) and also revealing the role in protein and starch metabolisms during grain filling under high day time temperature (HDT) stress (Payne et al. 2023).Four Osa-miR11343 genes found in rice HKT genes are involved in biotic stress by targeting the MLO domaincontaining protein (LOC_Os10g39520) in rice.One novel Osa-miR5819 has three target sites found in rice HKT gene-targeted CPuORF-containing bZIP38 TF and lipid transfer protein (LTPL118) subject to translational control via regulation by sucrose (Baldrich et al. 2015).Sucrose is a signal molecule that is involved in the activation of plant defense mechanisms.Another miR444 found in the rice HKT gene specifically targets the MADS-box transcription factors, which play an essential role in the HDT-induced caryopsis development, (Payne et al. 2023).Evidence has also shown that heat stress has been demonstrated to induce an upregulation of miR444 in maize (He et al. 2019).Interestingly, we found two microRNAs, Osa-miR1846 and Osa-miRN45, have several target sites in rice HKT genes.In both CDT and HDT, Osa-miR1846 was found to be strongly expressed during grain filling in the spikelets and its targeting of a heat shock factor (HSF) (Kushawaha et al. 2021).HSFs may cause chalkiness by increasing the expression of heat shock proteins (Kaneko et al. 2016).Therefore, it is probable that increased amounts of Osa-miR1846 in the Cypress inhibit this HSF, resulting in less chalkiness.On the other hand, Osa-miRN45 has a particular function to play throughout the differentiation process at the time of grain filling in rice (Peng et al. 2013).Together, these findings lay the groundwork for future genetic studies of OsHKT genes and facilitate the breeding of novel rice cultivars.

Conclusions
HKT family proteins are anticipated to be essential in plant salt stress tolerance.We extensively analyzed the HKT gene family in rice, both bioinformatically and functionally.This in silico investigation highlighted possible biological and molecular functions of the OsHKT genes in rice development and stress response.Phylogenetic and structural evaluations revealed that TrkH domains were highly significant for their respective roles.The rice HKT genes demonstrated purifying selection on chromosomes.Identification of cis-regulatory elements revealed their function in abiotic stress tolerance.Several transcription factors also modulate OsHKT gene expression to prevent salt toxicity in rice.OsHKT genes were found to be more active in roots and leaves under salt stress, suggesting they regulate rice plant growth, as revealed by tissue-specific expression studies.Our findings could help choose or target candidate genes for functional validation via molecular cloning in response to high salinity stress tolerance to improve crop plants.

Fig. 1
Fig. 1 Schematic representation of the steps taken to analyze the HKT family proteins in rice

Fig. 2
Fig. 2 The structure of the TrkH domain of HKT family proteins in Oryza sativa

Fig. 3
Fig. 3 Phylogenetic relationship (A) and schematic representation of the conserved motifs (B) of HKT family proteins in Oryza sativa

Fig. 5
Fig. 5 Chromosomal map and duplication event coordinates of HKT genes that are paralogous in Oryza sativa.The lines indicate the two pairs of paralogous genes presented in duplicated blocks, representing segmental duplication

Fig. 7
Fig. 7 Distribution of nine TFbs in rice HKT gene promoter regions.The pink and brown bars represent putative WRKY, bHLH, bZIP, MYB, AP2/ERF, GATA, B3, Dof, and C2H2 binding sites on the positive and negative strands of DNA, respectively

Fig. 8 AFig. 9
Fig. 8 A Distribution of TFbs and B Functional categorization of identified motifs in OsHKT gene promoter regions

Fig. 11
Fig. 11 Hierarchical clusters show expression patterns of OsHKT genes during the entire life cycle of rice (A) and under salinity (B).The color bar at the right represents the log 2 expression values: red, black, and green indicate high, medium, and low expression, respectively

Table 1
Characteristics of Rice HKT family gene was used to translate protein sequence.r indicates reverse, f indicates forward, bp indicates base pair, aa indicates amino acids, Da indicates Dalton, and pI indicates protein isoelectric point.Subcellular localization (SL) predictions: PM indicates plasma membrane *Nucleotide sequence

Table 2
Duplicated paralogous HKT gene pairs and their duplication time in Oryza sativa * Mya: Million years ago