Genome-Wide Analysis of the Oat (Avena sativa) HSP90 Gene Family Reveals Its Identification, Evolution, and Response to Abiotic Stress

Oats (Avena sativa) are an important cereal crop and cool-season forage worldwide. Heat shock protein 90 (HSP90) is a protein ubiquitously expressed in response to heat stress in almost all plants. To date, the HSP90 gene family has not been comprehensively reported in oats. Herein, we have identified twenty HSP90 genes in oats and elucidated their evolutionary pathways and responses to five abiotic stresses. The gene structure and motif analyses demonstrated consistency across the phylogenetic tree branches, and the groups exhibited relative structural conservation. Additionally, we identified ten pairs of segmentally duplicated genes in oats. Interspecies synteny analysis and orthologous gene identification indicated that oats share a significant number of orthologous genes with their ancestral species; this implies that the expansion of the oat HSP90 gene family may have occurred through oat polyploidization and large fragment duplication. The analysis of cis-acting elements revealed their influential role in the expression pattern of HSP90 genes under abiotic stresses. Analysis of oat gene expression under high-temperature, salt, cadmium (Cd), polyethylene glycol (PEG), and abscisic acid (ABA) stresses demonstrated that most AsHSP90 genes were significantly up-regulated by heat stress, particularly AsHSP90-7, AsHSP90-8, and AsHSP90-9. This study offers new insights into the amplification and evolutionary processes of the AsHSP90 protein, as well as its potential role in response to abiotic stresses. Furthermore, it lays the groundwork for understanding oat adaptation to abiotic stress, contributing to research and applications in plant breeding.


Introduction
The quality of life of terrestrial organisms on Earth is increasingly impacted by climate change [1].As sessile organisms, plants are particularly susceptible to environmental stresses such as drought, salinity, cold, and heat during growth and development [2].Recently, heat stress has become a significant abiotic stresses affecting normal plant growth and development due to global warming, increased droughts, and extreme weather conditions [3].In particular, prolonged growth at high temperatures severely inhibits starch synthesis and carbon assimilation, leading to a reduction in average yields and posing a major challenge to food security [4,5].Furthermore, the exposure of plants to premature high temperatures during unsuitable phenological periods makes them more susceptible to pathogen infection, potentially affecting the quality of crop production [6].
Plants have developed regulatory mechanisms to cope with heat stress and thousands of genes are involved during the evolution of their long-term adaptation [7].Among them, the heat shock protein (HSP) is one of the best-characterized genes and plays a significant role in regulating responses to heat stress.Generally, HSPs can be categorized into HSP20, HSP60, HSP70/DnaK, HSP90, and HSP100/ClpB families according to their molecular weight and sequence homogeneity [8], of which HSP90s are highly conserved in molecular evolution and an abundant family of ATP-dependent molecular chaperone proteins in prokaryotes and higher eukaryotes [9], which are broadly distributed in the cytoplasm, chloroplasts, mitochondria, and endoplasmic reticulum, accounting for 1-2% of total cellular proteins [10,11].HSP90 generally contains three structural domains: the N-terminal ATP-binding domain, the M domain, and the C-terminal substrate-binding domain [12].HSP90 features an unconventional Bergerat ATP-binding fold and is part of the GHKL superfamily [13].According to previous studies, HSP90 proteins often have dual functions: HSP90s are involved in regulating and maintaining the conformation of various proteins and assisting normal cell survival under stress on the one hand [14], and act as negative feedback regulators of heat stress responses on the other [15].HSP90, along with other molecular chaperones, provides a mechanism to promote protein folding [12], prevents protein aggregation, and facilitates the refolding of inactivated proteins, thereby increasing the resistance of certain cells [14].HSP90 expression is up-regulated when plants are stressed; it then associates with nonprotein substances to enable the repair of deformed proteins [16].Besides its importance for protein folding, a function of HSP90s as negative regulators for heat stress transcription factors (Hsfs) has been proposed for Hsf1, whose activities in facilitating downstream gene expression are tightly regulated [7].
Recently, HSP90s have been identified and found in many plants, including seven members of the HSP90 members in Arabidopsis thaliana [17], eight in Brachypodium distachyon [18], twenty-one in Nicotiana tabacum [19], and eight in Perennial Ryegrass [20], and directly or indirectly implicated in a host of physiological processes ranging from plant growth and development to abiotic and biotic stress responses [8].Overexpression of AtHSP90-2, AtHSP90-5, and AtHSP90-7 in Arabidopsis thaliana reduces tolerance to salt and drought stress but increases tolerance to high Ca 2+ concentrations [21].Both tobacco and Arabidopsis species have HSP90 members that convey resistance to pathogens by counteracting the response of signaling receptor R proteins from pathogens [22,23].Additionally, up-regulation of the VvHsp901a gene was delayed when grape plants were subjected to drought and high-temperature stresses [24].The HSP90 protein also plays a role in plant growth and developmental processes, and its mediated distribution of PIN1 regulates the distribution of auxin signaling, thereby promoting plant growth and development [25].
Oats (Avena sativa L.) are a global crop and one of the richest sources of protein, fat, and β-glucan among all cereals, with a low carbon footprint [26,27].Also, oats are a widely grown annual forage worldwide and an important source of high-quality pasture for livestock [26].Oats are a cool-season crop that is suitable for growing in humid environments [28], but due to severe global warming, temperature conditions are quickly met, which may lead to shorter growing periods, smaller plants, and lower yields in oats.Therefore, it is of great significance to understand and reveal the molecular members (e.g., HSP90s) that contribute to high temperature tolerance in oats.To date, a systematic and comprehensive study of the HSP90 gene family in oats has not yet been reported, and the assembly of a high-quality oat genome provides the necessary information to characterize HSP90s at the genome-wide level [29].In this study, we characterized the HSP90 gene within the oat genome, including gene sequence and homology analyses, and described the evolutionary pathway of the HSP90 gene in oats.Additionally, we analyzed oat HSP90 gene expression under high-temperature, salt, cadmium (Cd), polyethylene glycol (PEG), and abscisic acid (ABA) stresses using Quantitative Real-Time PCR.Our study provides a new avenue for molecular breeding in oats, contributes to a better understanding of the heat tolerance response of oats under high-temperature conditions, and lays the foundation for future studies on the function of the AsHSP90 protein in oat stress tolerance.

Identification of Oat HSP90 Genes and Chromosomal Distribution
A total of 20 HSP90 gene family members has been identified in A. sativa (Table 1).AsHSP90 proteins were renamed AsHSP90-1 to AsHSP90-20 based on their molecular weight.The obtained AsHSP90 sequences varied in length ranging from 627 to 809 amino acids, with pI values ranging from 4.6 to 5.09 and molecular weights ranging from 71,881.4 kd to 92,623.6 kd.AsHSP90 proteins are predominantly cytoplasmic and chloroplastic, except for AsHSP90-5, AsHSP90-10, AsHSP90-1, AsHSP90-15, and AsHSP90-20, which are localized in the nucleus and ER, respectively.Members of the AsHSP90 gene family are distributed across nine chromosomes and most of them are located in positions of high gene density (Figure 1), and most AsHSP90 genes are located on chr5 (chr5A, chr5C, chr5D).chr5 contains the most AsHSP90 genes, although it is not the longest chromosome.Gene clusters can be observed on chr5A, chr5C.This uneven distribution might be the result of uneven replication of oat chromosome segments.In addition, it was noted that relatively less AsHSP90 genes were located on chr2A, chr6A, and chr6C.

Phylogenetic Analysis of AsHSP90 Genes
In order to understand the evolutionary relationships of the HSP90 members, a phylogenetic tree including seven Arabidopsis HSP90s, eight rice HSP90s, eight Zea mays HSP90s, nine Brachypodium disachyon HSP90s, seven A. insularis HSP90s, and thirteen A.  .Phylogenetic tree analysis of the HSP90 proteins from A. sativa, A. insularis, A. longiglumis, Arabidopsis thaliana, Brachypodium disachyon, rice, and maize.The HSP90s were divided into six clades (Clades 1-6) based on the clustering of the protein sequence.The proteins from A. sativa, A. insularis, A. longiglumis, Arabidopsis thaliana, Brachypodium disachyon, rice, and maize are presented in brown, blue, dark orange, dark blue, green, gray, and dark green, respectively.

Motif Pa ern and Gene Structure Analyses of AsHSP90 Members
The evolution of the oat HSP90 gene family was revealed by analyzing the gene structure and motifs of the AsHSP90 genes.The 20 AsHSP90s proteins could be placed in six groups according to a constructed simplified phylogenetic tree.Of these, Groups 3 and 6 had the most and least members, with six and two, respectively (Figure 3A).All of these genes had between 2 and 18 introns.However, Groups 4, 5, and 6 contained 2 introns,

Motif Pattern and Gene Structure Analyses of AsHSP90 Members
The evolution of the oat HSP90 gene family was revealed by analyzing the gene structure and motifs of the AsHSP90 genes.The 20 AsHSP90s proteins could be placed in six groups according to a constructed simplified phylogenetic tree.Of these, Groups 3 and 6 had the most and least members, with six and two, respectively (Figure 3A).All of these genes had between 2 and 18 introns.However, Groups 4, 5, and 6 contained 2 introns, while the remaining three groups contained 15-18 introns (Figure 3C).In addition, genes on the same branch of the evolutionary tree are similar in structure, and their CDSs have similar numbers of introns.There was little variation in the location and length of the introns within the groups, but significant variation between the groups.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 6 of 19 contained 12-50 amino acids.Among them, motif 1 had the lowest amino acid content, with 12 amino acids.In addition, most of the genes consisted of 10 conserved motifs.Similar genes had similar motifs, which suggests that the AsHSP90 gene family has similar functions.Overall, the HSP90 gene family in oats is highly conserved, with few conserved motifs lost during evolution.

Duplication Analysis of AsHSP90 Members
Gene duplication events in the AsHSP90 gene were analyzed using MCScanX [31].In total, there were 10 pairs of duplication genes among HSP90 genes (Figure 4 and Table 2).All duplicates were from Group 3 and Group 4, and the majority of them were located at the end of the chromosome.These 10 pairs of genes were defined as WGD/segmental duplicates and inter-chromosomal.Some AsHSP90 genes had undergone more than one duplication.In addition, we calculated the non-synonymous (Ka) and synonymous (Ks) substitution rates, as well as Ka/Ks ratios (Table 2), to capture the evolutionary dynamics of the ASHSP90 protein coding sequence.Of these, 10 pairs of duplication genes had Ka/Ks values of <1, suggesting that these genes had undergone primarily purifying selection.To better understand the structural quality of the AsHSP90 protein, we identified 10 conserved motifs in the protein using the MEME [30] motif search tool and explored the distribution of these conserved motifs in the AsHSP90 protein (Figure 3B, Supplementary Figure S1).The results of this study showed that the 10 most conserved motifs detected contained 12-50 amino acids.Among them, motif 1 had the lowest amino acid content, with 12 amino acids.In addition, most of the genes consisted of 10 conserved motifs.Similar genes had similar motifs, which suggests that the AsHSP90 gene family has similar functions.Overall, the HSP90 gene family in oats is highly conserved, with few conserved motifs lost during evolution.

Duplication Analysis of AsHSP90 Members
Gene duplication events in the AsHSP90 gene were analyzed using MCScanX [31].In total, there were 10 pairs of duplication genes among HSP90 genes (Figure 4 and Table 2).All duplicates were from Group 3 and Group 4, and the majority of them were located at the end of the chromosome.These 10 pairs of genes were defined as WGD/segmental duplicates and inter-chromosomal.Some AsHSP90 genes had undergone more than one duplication.In addition, we calculated the non-synonymous (Ka) and synonymous (Ks) substitution rates, as well as Ka/Ks ratios (Table 2), to capture the evolutionary dynamics of the ASHSP90 protein coding sequence.Of these, 10 pairs of duplication genes had Ka/Ks values of <1, suggesting that these genes had undergone primarily purifying selection.

Collinearity Analysis of HSP90 Genes
To explore the evolutionary relationship between the oat AsHSP90 gene and other species, synteny analyses were performed on four representative plants, including two possible ancestral species of oats (A.insularis and A. longiglumis) and two monocotyledons (Oryza sativa and Brachypodium distachyon).Thirty-seven and nineteen AsHSP90 syntenic gene pairs were identified in A. insularis and A. longiglumis, respectively, and fifteen syntenic gene pairs each were identified in Oryza sativa and Brachypodium distachyon (Figure 5A, Supplementary Table S1).Nine AsHSP90s (AsHSP90-2, AsHSP90-3, AsHSP90-4, AsHSP90-12, AsHSP90-13, AsHSP90-14, AsHSP90-15, AsHSP90-16, and AsHSP90-17) are present as syntenic genes in these four species (Figure 5B).In addition, syntenic genes of the oat HSP90 gene Group 1 (AsHSP90-18, AsHSP90-19, and AsHSP-20) were not found in either rice or Brachypodium distachyon, but their respective syntenic genes were found in A. Insularis and A. longiglumis.On this basis, we separately detected AsHSP90 orthologs in rice, A. longiglumis, Brachypodium distachyon, and A. insularis with OrthoFinder (Supplementary Table S2). A. longiglumis and A. insularis contained 7 and 12 orthologous genes, respectively, and Brachypodium distachyon contained 3. Interestingly, two and one genes in A. insularis and A. longiglumis, respectively, showed to be orthologous to oat AsHSP90 gene Group 1 (AsHSP90-18, AsHSP90-19, and AsHSP-20), and all of these genes were from different sub-genomes.In addition, all three genes belong to the same group and none of them were involved in duplication events, which may suggest that these three genes are more conserved during the evolutionary process.

Cis-Element Analysis of AsHSP90 Gene Promoters
Sequences 1500 bp upstream of the promoter of each AsHSP90 gene were isolated from the oat genome, and cis-acting elements were predicted using PlantCARE [32] to characterize the expression of each AsHSP90 gene.A total of 32 cis-acting elements were analyzed (Figure 6, Supplementary Table S3).AsHSP90-18 has the highest number of homeopathic acting elements and AsHSP90-16 has the lowest number of homeopathic acting elements.The 20 AsHSP90 promoter regions involved hormone-responsive elements, light-responsive elements, environment-responsive elements, and developmentally relevant elements (Figure 6B).These elements mainly responded to hormonal and abiotic stresses.Interestingly, there are two cis-acting regulatory elements involved in circadian control in AsHSP90-3 and AsHSP90-6.Furthermore, a total of five cis-elements related to salicylic acid responsiveness were found in AsHSP90-14, AsHSP90-16, and AsHSP90-20.

Cis-Element Analysis of AsHSP90 Gene Promoters
Sequences 1500 bp upstream of the promoter of each AsHSP90 gene were isolated from the oat genome, and cis-acting elements were predicted using PlantCARE [32] to characterize the expression of each AsHSP90 gene.A total of 32 cis-acting elements were analyzed (Figure 6, Supplementary Table S3).AsHSP90-18 has the highest number of homeopathic acting elements and AsHSP90-16 has the lowest number of homeopathic acting The 20 AsHSP90 promoter regions involved hormone-responsive elements, light-responsive elements, environment-responsive elements, and developmentally relevant elements (Figure 6B).These elements mainly responded to hormonal and abiotic stresses.Interestingly, there are two cis-acting regulatory elements involved in circadian control in AsHSP90-3 and AsHSP90-6.Furthermore, a total of five cis-elements related to salicylic acid responsiveness were found in AsHSP90-14, AsHSP90-16, and AsHSP90-20.

Expression Analysis of AsHSP90s in Oats under Five Abiotic Stresses
To analyze the expression pa ern of AsHSP90 under several different abiotic stresses, 20 AsHSP90 proteins were analyzed using qRT-PCR.As shown in Figure 7, different expression pa erns of 20 AsHSP90s were observed under heat, drought, salt, Cd, and ABA

Expression Analysis of AsHSP90s in Oats under Five Abiotic Stresses
To analyze the expression pattern of AsHSP90 under several different abiotic stresses, 20 AsHSP90 proteins were analyzed using qRT-PCR.As shown in Figure 7, different expression patterns of 20 AsHSP90s were observed under heat, drought, salt, Cd, and ABA stresses.Almost all AsHSP90 members were involved in expression under different abiotic stresses.Among the five treatments, heat stress elicited the most pronounced stress response, with the average expression of AsHSP90 genes being two-eight times higher than that under the other four abiotic stresses.AsHSP90-7, AsHSP90-8, and AsHSP90-9 exhibited prominent expression in response to heat stress, while other members also demonstrated varying levels of transcriptional activation.
stresses.Almost all AsHSP90 members were involved in expression under different abiotic stresses.Among the five treatments, heat stress elicited the most pronounced stress response, with the average expression of AsHSP90 genes being two-eight times higher than that under the other four abiotic stresses.AsHSP90-7, AsHSP90-8, and AsHSP90-9 exhibited prominent expression in response to heat stress, while other members also demonstrated varying levels of transcriptional activation.Under the drought treatment (PEG treatment simulation), AsHSP90-12 and AsHSP90-13 were significantly expressed at 12 h, but their expression decreased with increasing exposure time.AsHSP90-1, AsHSP90-5, and AsHSP90-6 were also observed to be Under the drought treatment (PEG treatment simulation), AsHSP90-12 and AsHSP90-13 were significantly expressed at 12 h, but their expression decreased with increasing exposure time.AsHSP90-1, AsHSP90-5, and AsHSP90-6 were also observed to be expressed at the 6 h point.Furthermore, the expression of AsHSP90-3, AsHSP90-4, AsHSP90-5, AsHSP90-6, AsHSP90-10, and AsHSP90-20 was significantly reduced at 48 h.Under NaCl treatment, the expression of all members except AsHSP90-3 increased at 6 h and then decreased and then increased with longer exposure time.
Under Cd treatment, AsHSP90-13, AsHSP90-15, and AsHSP90-18 were significantly expressed at 12 h, and AsHSP90-2 was significantly expressed at 48 h.The expression of other AsHSP90s showed a fluctuating pattern, suggesting that their expression may be influenced by the duration of Cd treatment.AsHSP90-7, AsHSP90-9, and AsHSP90-10 were significantly expressed under ABA treatment at 6 h; AsHSP90-4 and AsHSP90-5 were significantly up-regulated at 48 h and continued to be up-regulated, while the expression of the other members was not significant.
In general, Group 5 (AsHSP90-7, AsHSP90-8, and AsHSP90-9) in the oat AsHSP90 gene was induced under all five stresses.Among all of the abiotic stresses, the highest expression of AsHSP90-9 was found after 12 h of heat stress.

Three-Dimensional Structure Prediction and Protein-Protein Interaction Network
Three-dimensional protein structures of the AsHSP90s were performed by the SWISS-MODEL [33] server and model generation was performed via PyMOL (Supplementary Figure S2).All targets had greater than 30% identity with the template, which is a threshold that is a sign of successful modeling (advances in homology protein structure modeling).
The QMEAN score values of the models varied between 0.64 and 0.76, which indicated that all of the models were of better quality, while the GMQE values ranged from 0.54 to 0.76.Meanwhile, out of the 20 models, 16 models were hetero-trimer states (Supplementary Table S4).
The protein-protein interaction network was further analyzed to detect interactions between AsHSP90 and related proteins.Seven other proteins were found to interact with AsHSP90 (Supplementary Figure S3), potentially being jointly involved in certain biological processes.Protein nodes were manually rearranged based on their degree of interaction.Proteins positioned in the inner circle of the layout exhibit a higher degree of interaction.In addition to AsHSP90, proteins A0A3B5YXX4 (HSP70), A0A3B6EMP3 (calreticulin), and A0A3B6H015 (calreticulin) also exhibit strong associations with AsHSP90 proteins.

Discussion
Heat stress protein 90 (HSP90) is a highly conserved molecular chaperone within the HSP family.HSP90 proteins are rapidly synthesized in response to heat stress treatments, serving to counteract the damage caused to plants by high temperatures [10].Here, we identified twenty HSP90 genes and assigned them to six clades.Furthermore, the number of oat HSP90 genes was higher than in some previously studied species, such as the 7 in A. thaliana [17] and 10 in populus [34], a phenomenon largely attributable to hexaploidy.These AsHSP90 genes may play a significant role in the physiological maintenance of oats, allowing them to survive high temperatures and other abiotic stress environments.The HSP90 family of proteins exhibits varied biophysical properties, indicative of a wide diversity among its members.This diversity lays the groundwork for further studies on the function of HSP90 genes.In this study, the AsHSP90 gene sequence displayed an isoelectric point (pI) ranging from 4.60 to 5.09 and was acidic, consistent with previous studies on the HSP90 gene in perennial ryegrass [20].Additionally, we observed that the AsHSP90 gene is unevenly distributed in oats, predominantly in regions of high gene density on the chromosomes.This distributional characteristic may be linked to the uneven replication of oat chromosome segments [29].
Oats undergo complex polyploidization events and frequent translocations among subgenomes, which provide a good opportunity to study gene family formation and expansion [29,35].Gene duplication events are thought to be an important mechanism for increasing gene family diversity [36].Plant evolution is usually accompanied by gene fragment duplication events, considered as one of the main drivers of the expansion of plant gene families.The oat genome possesses a mosaic chromosome structure, and the chromosomal rearrangements it undergoes often result in the duplication of gene family members [29].In this study, 10 pairs of segmental duplication genes were identified in the AsHSP90 gene family.These genes belong to Group 3 and Group 4, comprising homologous genes with similar structural domains.Chromosomal polyploidy also significantly contributes to the expansion of the number of gene families in plants.The "A" subgenome and "CD" subgenome in oats originated from A. insularis and A. longiglumis, respectively [35].Also, 13 and 7 HSP90 genes were identified from A. insularis and A. longiglumis, the probable ancestral species of oats, respectively, and the total number of these genes is consistent with the number of AsHSP90 genes.Thus, chromosome doubling is likely responsible for AsHSP90 amplification in oats.This finding is consistent with the fact that A. insularis and A. longiglumis hybridized to form a heterozygous hexaploid.Furthermore, synteny analysis and orthologous gene identification reveal that not all oat AsHSP90 genes have direct homologs in the ancestral species.For example, the AsHSP90-9 gene located in the "A" subgenome lacks orthologous genes in A. insularis, whereas AsHSP90-7 and AsHSP90-8, which are part of Group 3 in the "CD" subgenome, possess orthologous genes in A. longiglumis (Supplementary Table S2).Additionally, there is a gene duplication event between AsHSP90-7, AsHSP90-8, and AsHSP90-9, suggesting that AsHSP90-9 may be a paralogous gene resulting from this duplication.This implies that the formation of paralogous genes also contributes to the expansion of the AsHSP90 gene family.In conclusion, the formation of the AsHSP90 gene family in oats may have arisen mainly through gene duplication after polyploidy and divergence.
Phylogenetic analyses can help to understand evolutionary relationships between species and ermine homology between and within species [37].In this study, a phylogenetic tree was constructed using the protein sequences of the HSP90 gene from seven monocotyledonous and one dicotyledonous plant species.Based on the phylogenetic analysis, it can be categorized into six distinct clades, wherein all Arabidopsis HSP90 genes cluster in Clade 5, which may be attributed to Arabidopsis being the sole dicotyledonous plant in the phylogenetic tree [38].Furthermore, the phylogenetic tree also reflects the closer relationship of oats and A. insularis to A. longiglumis.Intron gain or loss and intron density significantly impact the evolution of large eukaryotic genomes.According to the phylogenetic tree, the AsHSP90 gene is categorized into six groups, each with a highly similar exon number and exon-intron structure.Similarly, the motif distribution of AsHSP90 genes across various subgroups exhibits a consistent pattern.Consequently, these analyses further corroborate the reliability of this phylogenetic classification of AsHSP90 genes.
Numerous studies have demonstrated that the plant HSP90 protein plays a crucial role in responding to various abiotic stresses [39,40].According to the qRT-PCR results, it was observed that most AsHSP90 genes were induced under all five stresses, albeit with low expression levels in some genes.HSP is a well-known protein that responds to heat stress and protects plants from high-temperature stress damage [41].In the current study, the highest expression of all AsHSP90 proteins was observed under high-temperature stress, suggesting that HSP90 is particularly sensitive to heat stress.Group 5 (AsHSP90-7, AsHSP90-8, and AsHSP90-9) within the oat AsHSP90 gene family exhibited high expression under all five stress conditions.Among them, the AsHSP90-9 gene, a paralogous gene of AsHSP90-7 and AsHSP90-8 replicated, had the highest expression under heat stress.Gene duplication events usually result in an increase in the number of genes; after this, the replicated genes may undergo neofunctionalization or subfunctionalization [42].In our study, AsHSP90-9 had similar expression patterns to AsHSP90-7 and AsHSP90-8, and we hypothesized that the AsHSP90-9 gene may undergo subfunctionalization to carry out some of the functions of the AsHSP90-7 and AsHSP90-8 genes.Therefore, under hightemperature stress conditions, the expression of the AsHSP90-9 genes involved in the response to heat stress after subfunctional differentiation was significantly up-regulated to help the oats better adapt to heat stress.Furthermore, the analysis of the cis-elements of AsHSP90 in this study revealed a variety of cis-acting elements involved in hormone regulation and abiotic stress.For instance, AsHSP90-18, AsHSP90-19, and AsHSP90-19 had the highest number of ABRE-binding sites within the ABA regulatory pathway.ABAregulated genes are involved in multiple biotic and abiotic stress responses in plants [43].However, contrary to expectations, these genes did not exhibit the highest expression levels in our RT-qPCR analysis under the five abiotic stress treatments.This discrepancy might be attributed to their status as orthologous genes from different subgenomes, suggesting that their expression is regulated through a complex mechanism of subgenomic homologous co-expression.Thus, the roles of AsHSP90-9, AsHSP90-18, AsHSP90-19, and AsHSP90-20 in response to these stresses warrant further investigation.Moreover, variations were observed in gene expression at different sites, necessitating further studies to investigate the expression of the AsHSP90 protein in various tissues of oats.

HSP90 Gene Family Identification in Avena sativa, Avena insularis, and Avena longiglumis
The genomic resources of Avena sativa and its possible ancestors Avena insularis and Avena longiglumis were cv "sang_v1.1",cv "BYU209_v1.1", and cv "CN58138_v1.1"from the GrainGenes database (https://wheat.pw.usda.gov,accessed on 24 May 2023) [44], and the Hidden Markov Model (HMM) matrix file for the HSP90 structural domain was obtained from the Pfam database (PF00183).Using HMMER software version 3.0 [45], HSP90s were searched for within three genome protein sequences.A BLASTP analysis of these three genomes' genomic resources was conducted using the protein sequence of the Brachypodium disachyon and Arabidopsis thaliana HSP90 gene as a query.Based on the HMMER and BLASTP results, all candidate HSP90 proteins that possibly contained the HSP90 domain were submitted to Pfam (http://pfam.xfam.org/,accessed on 24 May 2023) and CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi,accessed on 24 May 2023) for confirmation of the final resulted HSP90 members.A. sativa HSP90 genes were renamed according to their molecular weight.An in-house Perl script was used to calculate the molecular weight, instability index, and theoretical isoelectric point (pI) of AsHSP90.Subcellular localization was predicted using WoLF PSORT [46] (https: //wolfpsort.hgc.jp/accessed on 29 May 2023).All HSP90 genes were mapped to A. sativa chromosomes using TBtools-II [47].

Phylogenetic Analysis of HSP90 Genes
The HSP90 protein sequences of Avena sativa, Avena insularis, Avena longiglumis, Arabidopsis thaliana, Oryza sativa, Brachypodium disachyon, and Zea mays were subjected to multiple-sequence alignment analysis using ClustalW [48] in order to study the evolutionary relationship between the HSP90 families of these five plants.A maximum-likelihood (ML) phylogenetic tree was construction by 1000 bootstrapping with MEGA [49].

Structure and Conserved-Motif Analysis of AsHSP90s
To extract the CDS and UTR locations corresponding to AsHSP90s, we used in-house Perl scripts.In addition, in the AsHSP90 protein, a phylogenetic tree was constructed using the maximum-likelihood (ML) method and 10 motifs were identified using the MEME program 5.10 [30] (https://meme-suite.org/meme/tools/fimo, accessed on 16 June 2023).The gene structures, motifs, and phylogenetic tree were mapped and modified using TBtools-II [47].

Gene Duplication and Ka and Ks Calculation
Gene duplication search for the identified HSP90 members was performed using blastall.The major criteria used for analyzing potential gene duplications included: (a) length of alignable sequence covers >75% of longer gene, and (b) similarity of aligned regions >75% [50].The rate of synonymous substitutions (Ka) and nonsynonymous substitutions (Ks) in the AsHSP90 gene obtained from gene duplication events was calculated using KaKs_Calculator 3.0 [51].

Synteny Analysis of AsHSP90 Genes and Selected Plants
To demonstrate the synteny relationship of the HSP90 genes obtained from Avena sativa and gramineous species of Oryza sativa, Brachypodium disachyon, A. insularis, and A. longiglumis, the syntenic analysis maps were constructed using the software MCScanX [31].In addition, gene duplication events in AsHSP90s of oats also were visualized with MC-ScanX.OrthoFinder 2.5.5 [52] was utilized to find directly orthologous genes between the oats and other representative species.

Identification and Analysis of cis-Elements in the Promoter Region of AsHSP90
An in-house Perl script was used to extract 1.5 kb sequences upstream of the AsHSP90 gene as promoter regions and submit these sequences to PlantCARE [32] for the analysis of cis-regulatory elements.

Three-Dimensional Protein Structure Prediction
Protein templates in the PDB database [53] (https://www.rcsb.org/,accessed on 21 June 2023) with similar 3D structures to the AsHSP90 protein were searched using PSI-BLAST [54], and the resulting templates and AsHSP90 protein sequences were submitted to SWISS-MODEL [33] (https://swissmodel.expasy.org/,accessed on 22 June 2023) for protein 3D structure prediction.Moreover, the 3D model's quality was assessed with global model quality estimates (GMQE) and QMEAN values.The GMQE scores ranged from 0 to 1, with higher scores indicating a more reliable model, and the QMEAN scores ranged between 0 and −4, with models closer to 0 being of better quality.

Protein-Protein Interaction Network
To predict the interactions between oat HSP90 proteins and related proteins, the AsHSP90 protein sequence was submitted to the STRING database [55].The organism was set to wheat, and advanced settings were maintained at their default values.PPI networks were visualized using Cytoscape v3.10.1 [56].

Plant Material, Growth Conditions, and Treatment
Viable seeds of oat cv "Baylor" were grown in quartz sand and grown in a greenhouse.The germinated seedlings were transferred into Hoagland's solution after 7 days.Seedlings were subjected to stress treatments after 14 days.Salt stress was simulated by dissolving a 250 mM concentration of NaCl in the culture broth.Chromium (Cr) (K 2 Cr 2 O 7 ) was dissolved at a concentration of 300 mg/L to simulate heavy-metal stress.For heat stress, the temperature was 40 • C during the day/30 • C at night, and the photoperiod was 12 h of light/12 h of dark.Drought stress was stimulated with 20% polyethylene glycol 6000 (PEG) after lysis.Abscisic acid (ABA) was sprayed at a concentration of 100 mM.Live biological replicates of plant leaf samples were collected at 0 h, 3 h, 6 h, 12 h, and 48 h after each application of stress.Then, the stressed seedlings were collected for RNA extraction and stored at a temperature of −8 • C. 4.10.RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR Expression Analyses Total RNA was isolated using the Direct-zol™ RNA MiniPrep Kit (Zymo Research, Beijing, China), according to the manufacturer's protocol.ABScript III RT Master Mix for qPCR with Gdna Remover (Abclonal, Wuhan, China) was used for the synthesis of cDNA.RT-qPCR analyses were performed using the Genious 2× SYBR Green Fast Qpcr Mix (Abclonal, China), in accordance with the manufacturer's protocol; the reactions were run using the CXF96 Connect™ Real-Time System (Bio-Rad, Singapore).The AsEIF4A gene was used as an internal reference gene to calculate the expression of 20 AsHSP90 genes [57].Relative expression was calculated using the 2 −∆∆Ct method [58,59].In addition, twenty primer pairs for the AsHSP90 gene were designed with Primer 5 software.Primers for the AsHSP90 gene used in the qRT-PCR assay are listed in Supplementary Table S5.

Conclusions
We identified and localized 20 AsHSP90 genes in the oat genome and divided them into six clades.Gene structures and motifs were highly conserved within the same groups.The formation of the AsHSP90 gene family in oats may have arisen mainly through gene duplication after polyploidy and divergence.Under high-temperature, salt, cadmium (Cd), polyethylene glycol (PEG), and abscisic acid (ABA) stresses, AsHSP90 showed the strongest expression under heat stress, and members of Group 5 (AsHSP90-7, AsHSP90-8, and AsHSP90-9) were generally highly expressed.The function of AsHSP90 proteins remains unknown, especially the co-expression of homologous proteins among different subgenomes, and further studies are needed to determine their precise function.Our study elucidates the potential pathways for the expansion of the AsHSP90 gene family in oats, and also lays the foundation for future functional analyses of these AsHSP90 proteins as well as studies of their synergistic expression across subgenomes.

Figure 1 .
Figure 1.Chromosomal localization of members of the oat HSP90 gene family.Blue to yellow colors within the chromosomes indicate increased gene density.Chromosome numbers are shown at the right of the vertical bar; gene locations are shown at the left of the vertical bar.

Figure 1 .
Figure 1.Chromosomal localization of members of the oat HSP90 gene family.Blue to yellow colors within the chromosomes indicate increased gene density.Chromosome numbers are shown at the right of the vertical bar; gene locations are shown at the left of the vertical bar.

Figure 2
Figure 2. Phylogenetic tree analysis of the HSP90 proteins from A. sativa, A. insularis, A. longiglumis, Arabidopsis thaliana, Brachypodium disachyon, rice, and maize.The HSP90s were divided into six clades (Clades 1-6) based on the clustering of the protein sequence.The proteins from A. sativa, A. insularis, A. longiglumis, Arabidopsis thaliana, Brachypodium disachyon, rice, and maize are presented in brown, blue, dark orange, dark blue, green, gray, and dark green, respectively.

Figure 2 .
Figure 2. Phylogenetic tree analysis of the HSP90 proteins from A. sativa, A. insularis, A. longiglumis, Arabidopsis thaliana, Brachypodium disachyon, rice, and maize.The HSP90s were divided into six clades (Clades 1-6) based on the clustering of the protein sequence.The proteins from A. sativa, A. insularis, A. longiglumis, Arabidopsis thaliana, Brachypodium disachyon, rice, and maize are presented in brown, blue, dark orange, dark blue, green, gray, and dark green, respectively.

Figure 3 .
Figure 3. Phylogenetic tree, motif analysis, and gene structure of AsHSP90: (A) Phylogenetic tree analysis of the AsHSP90 protein.(B) Motif composition of AsHSP90.(C) Gene structure of the AsHSP90 genes in oats.

Figure 3 .
Figure 3. Phylogenetic tree, motif analysis, and gene structure of AsHSP90: (A) Phylogenetic tree analysis of the AsHSP90 protein.(B) Motif composition of AsHSP90.(C) Gene structure of the AsHSP90 genes in oats.

Figure 4 .
Figure 4. Gene duplications of the oat HSP90 gene family.Red lines indicate duplicated gene pairs in AsHSP90, and gray lines indicate co-linear gene pairs in the genome.

Figure 4 .
Figure 4. Gene duplications of the oat HSP90 gene family.Red lines indicate duplicated gene pairs in AsHSP90, and gray lines indicate co-linear gene pairs in the genome.

Figure 5 .
Figure 5. Synteny analysis of AsHSP90s in A. sativa and four representative plants.(A) All AsHSP90 synteny genes in oats and in Oryza sativa, A. longiglumis, Brachypodium distachyon, and A. insularis are indicated by red lines.The synteny blocks in the oats and the other species are shown in gray lines.(B) The nine AsHSP90 genes with covariance in the four species are shown as purple, blue, yellow, and green lines.

Figure 5 .
Figure 5. Synteny analysis of AsHSP90s in A. sativa and four representative plants.(A) All AsHSP90 synteny genes in oats and in Oryza sativa, A. longiglumis, Brachypodium distachyon, and A. insularis are indicated by red lines.The synteny blocks in the oats and the other species are shown in gray lines.(B) The nine AsHSP90 genes with covariance in the four species are shown as purple, blue, yellow, and green lines.

Figure 6 .
Figure 6.Distribution of cis-elements upstream of oat AsHSP90: (A) Distribution of types and numbers of cis-elements in the promoter of the oat AsHSP90 genes.(B) Classification and proportion of cis-elements in the AsHSP90 gene.

Figure 6 .
Figure 6.Distribution of cis-elements upstream of oat AsHSP90: (A) Distribution of types and numbers of cis-elements in the promoter of the oat AsHSP90 genes.(B) Classification and proportion of cis-elements in the AsHSP90 gene.

Figure 7 .
Figure 7.The relative expression of 20 AsHSP90 genes in oat leaves was detected with qRT-PCR after treatments of 0 h (CK), 3 h, 6 h, 12 h, and 48 h under different abiotic stresses.Error bars indicate the standard error (SE) between three replicates.

Figure 7 .
Figure 7.The relative expression of 20 AsHSP90 genes in oat leaves was detected with qRT-PCR after treatments of 0 h (CK), 3 h, 6 h, 12 h, and 48 h under different abiotic stresses.Error bars indicate the standard error (SE) between three replicates.

Table 1 .
Biophysical properties and subcellular localization of the oat HSP90 genes.