Genome-Wide Identification and Bioinformatics Analysis of the FK506 Binding Protein Family in Rice

The FK506 Binding Protein (FKBP), ubiquitously present across diverse species, is characterized by its evolutionarily conserved FK506 binding domain (FKBd). In plants, evidence suggests that this gene family plays integral roles in regulating growth, development, and responses to environmental stresses. Notably, research on the identification and functionality of FKBP genes in rice remains limited. Therefore, this study utilized bioinformatic tools to identify 30 FKBP-encoding genes in rice. It provides a detailed analysis of their chromosomal locations, evolutionary relationships with the Arabidopsis thaliana FKBP family, and gene structures. Further analysis of the promoter elements of these rice FKBP genes revealed a high presence of stress-responsive elements. Quantitative PCR assays under drought and heat stress conditions demonstrated that genes OsFKBP15-2, OsFKBP15-3, OsFKBP16-3, OsFKBP18, and OsFKBP42b are inducible by these adverse conditions. These findings suggest a significant role for the rice FKBP gene family in stress adaptation. This research establishes a critical foundation for deeper explorations of the functional roles of the OsFKBP genes in rice.


Introduction
FK506 Binding Proteins (FKBPs) are a class of cellular receptor proteins, widely present in organisms and categorized as immunophilins, which are capable of binding with immunosuppressive drugs such as Cyclosporine-A (CsA), FK506, and rapamycin [1].Since the 1980s, concerted efforts have enabled the successful isolation and purification of various FKBPs which specifically bind to FK506 and rapamycin.These interactions between FKBPs and immunosuppressants have revealed the significant potential of immunophilins in organ transplantation applications [2,3].
All members of the FKBP family contain an FK506 binding domain (FKBd), composed of approximately 110 amino acids.This region serves not only as the binding site for FK506 and rapamycin but also as the active site for peptidyl-prolyl cis-trans isomerase, resulting in a highly conserved amino acid sequence and tertiary structure for this domain [4][5][6].In the plant FKBP family, molecular weights of different members vary due to diverse structural compositions.For example, Arabidopsis AtFKBP12 and riceOsFKBP12 are composed solely of an FKBd domain and have a molecular weight of only 12 kDa, whereas rice OsFKBP75 and wheat wFKBP77 possess multiple domains and exceed a molecular weight of 70 kDa [7].Studies have also shown that in plants and other species, larger FKBP molecules include additional structural domains beyond the FKBd, such as Tetratricopeptide Repeat (TPR) and calmodulin-binding domains [8,9].For instance, Arabidopsis AtFKBP62 and AtFKBP65 can bind to the heat shock protein HSP90 through their TPR domain, thus regulating Arabidopsis'sheat tolerance [8][9][10][11].Research by Meiri [9,12] indicates that AtFKBP62 and AtFKBP65 influence the production of small heat shock proteins related to high-temperature stress repair, yet they exhibit opposite functions during prolonged high-temperature stress developmental processes in Arabidopsis.
Studies have highlighted the critical roles of FKBP gene family members in Arabidopsis growth, development, and stress responses.For instance, the Arabidopsis mutant fkbp42 of AtFKBP42 displays spiral root growth and an overall slow development, thus also known as TWD1 (TWISTED DWARF1).Further research has shown that TWD1 interacts with ATP Binding Cassette B auxin efflux transporter-associated proteins AtABCB1, AtABCB4, and AtABCB19, potentially affecting the cellular localization of these transport proteins and thereby disrupting root auxin transport, impacting plant growth and development [13].Arabidopsis AtFKBP72 is involved in the biosynthesis of Very Long-Chain Fatty Acids (VLCFAs); the disruption of its function interrupts VLCFA production, leading to abnormal cell division and resulting in the Arabidopsis mutant with the "PASTICCINO" phenotype [8].AtFKBP15-1 and AtFKBP5-2 inhibited lateral root development in Arabidopsis [10].In rice, OsFKBP20-1a and OsFKBP20-1b share 85% homology and are both expressed in the nucleus.Although their expression patterns differ across tissues, both are induced by high temperatures and drought.According to Ahn, OsFKBP20-1a is highly expressed in various plant tissues and is induced by high temperatures and drought stress [14].In contrast, the expression of OsFKBP20-1b begins to rise 24 h post-stress and is also present in the cytoplasm.OsFKBP20-1a can bind with SUMO (Small Ubiquitin like Modifier)-conjugated enzymes, and through SUMOylation, these enzymes can bind with other target proteins, thus participating in the high-temperature stress response; however, their specific functions and mechanisms of action have yet to be elucidated [15].
In wheat, FKBP genes are unevenly distributed across all 21 chromosomes, with their subcellular localization primarily in the nucleus, endoplasmic reticulum, cytoplasm, and chloroplasts [16].The FKBP gene family has also been identified in cucumbers, with 19 members predicted to be expressed in chloroplasts, the nucleus, cytoplasm, cytoskeleton, and peroxisomes [17].The FKBP gene family not only plays a vital role in the life processes of plants but it is also indispensable in animals.In studies on the Asian migratory locust (Locusta migratoria L.), researchers have identified 10 genes containing the FKBP_C (peptidyl-prolyl cis-trans isomerase) domain, which may play significant roles in embryo development and immune processes [18].
As a critical food crop and model plant, rice contains multiple genes encoding FKBPs, yet research on the identification and functionality of this gene family in rice is limited.This study, through bioinformatics methods, identified 30 genes encoding FKBP in rice (Oryza sativa L.) and analyzed their chromosomal locations, evolutionary relationships with the Arabidopsis family, gene structures, and promoter elements.This foundational study lays the groundwork for in-depth functional research on this family and the molecular breeding of rice for stress tolerance.

Identification of FKBP Genes
Rice genome sequences were retrieved from the Phytozome database (https://phytozomenext.jgi.doe.gov/(accessed on 9 May 2024), and amino acid sequences of Arabidopsis thaliana AtFKBP genes were sourced from the TAIR database (https://www.arabidopsis.org/index.jsp(accessed on 9 May 2024)) to serve as reference sequences.Local BLAST searches using TBtools were conducted to identify potential members of the rice OsFKBP gene family.The characteristic FKBP_C conserved domain (PF00254) associated with the FKBP gene family was obtained as a Hidden Markov Model from the InterPro database (https://www.ebi.ac.uk/interpro/entry/ pfam/ (accessed on 10 May 2024)), and searches were performed using HMMER3.0software to identify candidate members [19,20].These candidate members were then merged, and a reverse BLASTp analysis was conducted using the NCBI (National Center for Biotechnology) database (https://blast.ncbi.nlm.nih.gov/Blast.cgi(accessed on 11 May 2024)) to refine the selection of OsFKBP family genes further [21].Domain predictions and verifications were carried out using the NCBI CD-search tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi(accessed on 11 May 2024)), with any candidates missing domains or exhibiting redundant transcripts excluded from the final OsFKBP gene list.

Chromosomal Localization and Collinearity Analysis of FKBP Genes
The chromosomal localization of the OsFKBP family genes was visualized using TBtools v2.041 software based on annotated rice genome information.Collinearity analysis of rice OsFKBP genes and between rice and Arabidopsis FKBP genes was conducted using the One Step MCScanX-Super Fast component of TBtools v2.041 software [24].

Analysis of FKBP Gene Structures
Structural information for rice OsFKBP genes was extracted using TBtools v2.041 software for gene structure analysis.The MEME suite (https://meme-suite.org/meme/ tools/meme (accessed on 12 May 2024)) was utilized to analyze the conserved motifs within the OsFKBP sequences, with the motif count was set to 10 and all other parameters were at default settings.Structural domain predictions were conducted using the NCBI-CDD database (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi(accessed on 12 May 2024)) [25].The visualization of gene structures, conserved motifs, and structural domains was performed using TBtools v2.041 software.

Experimental Materials and Conditions
The experimental subject was the Japonica rice variety Nipponbare.Seedlings were cultured hydroponically: seeds were soaked until germination.After germination, seedlings were maintained in a rice nutrient solution [27].At 14 days post-germination, seedlings underwent stress treatment with 22% PEG 6000 and were subjected to a temperature of 45 • C under a light/dark cycle of 16/8 h with a light intensity of 200 mmol m −2 s −1 for 24 h.Aerial parts of the rice (including stems and leaves) were sampled at 0, 6, 12, and 24 h post-treatment and immediately frozen in liquid nitrogen.

Total RNA Extraction, Reverse Transcription, and Quantitative RT-PCR
Total RNA was extracted using the TRIzol method.Reverse transcription was performed using reverse transcription kits provided by Yeasen Biotech (Shanghai, China) Co., Ltd., and quantitative RT-PCR was conducted using Yeasen's Hieff ® qPCR SYBR Green Master Mix (11201ES08, Shanghai, China).Actin was used as the internal reference gene for expression analysis, conducted using a BIO RAD real-time fluorescence quantitative PCR instrument.The qPCR reaction volume was 10 µL, consisting of 5 µL of Hieff ® qPCR SYBR Green Master Mix (Shanghai, China), 0.2 µL each of forward and reverse primers (10 µM), 2 µL of cDNA template, and 2.6 µL of RNase-free H 2 O.The qPCR protocol included an initial denaturation at 95 • C for 5 min, followed by 40 cycles of denaturation at 95 • C for 10 s, annealing at 55-60 • C for 20 s, and extension at 72 • C for 20 s.The reaction was performed with biological triplicates as described previously [28,29].Relative gene expression was calculated using the formula: Relative Expression = 2 −∆∆CT .Primer sequences used for RT-PCR are listed in Table S2.
cluded an initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation a 95 °C for 10 s, annealing at 55-60 °C for 20 s, and extension at 72 °C for 20 s.The reactio was performed with biological triplicates as described previously [28,29].Relative gen expression was calculated using the formula: Relative Expression = 2 −ΔΔCT .Primer se quences used for RT-PCR are listed in Table S2.

Chromosomal Localization Analysis of FKBP Genes
Utilizing the annotation data from the rice genome, 30 rice OsFKBP genes were mapped to chromosomes using the TBtools software.A chromosomal distribution map was created for these genes (Figure 2).The localization analysis reveals that, with the exception of chromosome 10, which lacks any OsFKBP genes, and OsFKBP65d, which is unanchored to any chromosome, the other 29 OsFKBP genes are unevenly distributed across the remaining 11 chromosomes.Chromosome 2 harbors the largest number of OsFKBP genes, with six identified; followed by chromosome 1 with five; chromosome 9 with four; and chromosome 7 with three genes.Chromosomes 3, 4, 6, and 8 each contain two OsFKBP genes, whereas chromosomes 5, 11, and 12 each support only one OsFKBP gene.Additionally, OsFKBP62a, OsFKBP62b, and OsFKBP62c are tightly clustered on chromosome 1, yet collinearity analysis reveals no evolutionary duplication among them.
of the OsFKBP and AtFKBP families, and named rice OsFKBP family members based on the similarity of the AtFKBP family in Arabidopsis.The tree displays five phylogenetic subfamilies (numbered I to V and marked with different alternating colors).The OsFKBP family in rice was one where internal nodes may not necessarily provide true phylogenetic relationships between different offspring.

Chromosomal Localization Analysis of FKBP Genes
Utilizing the annotation data from the rice genome, 30 rice OsFKBP genes were mapped to chromosomes using the TBtools software.A chromosomal distribution map was created for these genes (Figure 2).The localization analysis reveals that, with the exception of chromosome 10, which lacks any OsFKBP genes, and OsFKBP65d, which is unanchored to any chromosome, the other 29 OsFKBP genes are unevenly distributed across the remaining 11 chromosomes.Chromosome 2 harbors the largest number of OsFKBP genes, with six identified; followed by chromosome 1 with five; chromosome 9 with four; and chromosome 7 with three genes.Chromosomes 3, 4, 6, and 8 each contain two OsFKBP genes, whereas chromosomes 5, 11, and 12 each support only one OsFKBP gene.Additionally, OsFKBP62a, OsFKBP62b, and OsFKBP62c are tightly clustered on chromosome 1, yet collinearity analysis reveals no evolutionary duplication among them.6), the number of identified FKBP distributed on this chromosome.

Collinearity Analysis of FKBP Genes
Using the One Step MCScanX-Super Fast module of TBtools software, collinear genomic regions were identified, and collinearity maps were constructed based on positional data both within rice and between rice and Arabidopsis (Figures 3 and 4).The analysis within species revealed four pairs of collinear genes (OsFKBP20-1a with OsFKBP20-1b, OsFKBP42a with OsFKBP42b, OsFKBP15-1 with OsFKBP15-2, and OsFKBP15-2 with OsFKBP16-3).This suggests that some members of the rice OsFKBP gene family might have originated from gene duplication events.The interspecific analysis showed that chromosomes 1 and 5 in rice are collinear with chromosome 3 in Arabidopsis, with two pairs of collinear genes identified between rice and Arabidopsis (OsFKBP20-1a with AtFKBP20-1, and OsFKBP20-1b with AtFKBP20-1).These findings imply a potential evolutionary homology between the Arabidopsis FKBP20-1 and rice FKBP20-1 genes.6), the number of identified FKBP distributed on this chromosome.

Collinearity Analysis of FKBP Genes
Using the One Step MCScanX-Super Fast module of TBtools software, collinear genomic regions were identified, and collinearity maps were constructed based on positional data both within rice and between rice and Arabidopsis (Figures 3 and 4).The analysis within species revealed four pairs of collinear genes (OsFKBP20-1a with OsFKBP20-1b, OsFKBP42a with OsFKBP42b, OsFKBP15-1 with OsFKBP15-2, and OsFKBP15-2 with OsFKBP16-3).This suggests that some members of the rice OsFKBP gene family might have originated from gene duplication events.The interspecific analysis showed that chromosomes 1 and 5 in rice are collinear with chromosome 3 in Arabidopsis, with two pairs of collinear genes identified between rice and Arabidopsis (OsFKBP20-1a with AtFKBP20-1, and OsFKBP20-1b with AtFKBP20-1).These findings imply a potential evolutionary homology between the Arabidopsis FKBP20-1 and rice FKBP20-1 genes.

Analysis of Conserved Motifs, Domains, and the Gene Structures of FKBPs
The conserved motifs and domains of the rice OsFKBP family were analyzed using the MEME Suite 5.5.5 online server and the NCBI-CDD online database.Based on the an notated rice genome data, a diagram of the conserved motifs, domains, and gene struc tures of the rice OsFKBPs was constructed (Figure 5).The analysis of conserved motifs revealed that each rice OsFKBP gene contains between one to ten motifs, with most con taining motifs 5, 7, 1, and 2. These motifs are highly conserved components within the FKBP_C domain, and their relative positions are largely similar across the family.The

Analysis of Conserved Motifs, Domains, and the Gene Structures of FKBPs
The conserved motifs and domains of the rice OsFKBP family were analyzed using the MEME Suite 5.5.5 online server and the NCBI-CDD online database.Based on the annotated rice genome data, a diagram of the conserved motifs, domains, and gene structures of the rice OsFKBPs was constructed (Figure 5).The analysis of conserved motifs revealed that each rice OsFKBP gene contains between one to ten motifs, with most containing motifs 5, 7, 1, and 2. These motifs are highly conserved components within the FKBP_C domain, and their relative positions are largely similar across the family.The analysis of conserved domains indicated that, apart from the OsTIG protein, which possesses a Tig domain, the remaining 29 rice OsFKBPs all contain the typical FKBP_C conserved domain characteristic of the FKBP gene family.The domain positions and compositions vary among different subgroups: subgroup I members have an NPL domain at the N-terminus and an FKBP_C domain at the C-terminus; subgroups II and III members have only the FKBP_C domain at the C-terminus; subgroup IV members have only the FKBP_C

Analysis of Conserved Motifs, Domains, and the Gene Structures of FKBPs
The conserved motifs and domains of the rice OsFKBP family were analyzed using the MEME Suite 5.5.5 online server and the NCBI-CDD online database.Based on the annotated rice genome data, a diagram of the conserved motifs, domains, and gene structures of the rice OsFKBPs was constructed (Figure 5).The analysis of conserved motifs revealed that each rice OsFKBP gene contains between one to ten motifs, with most containing motifs 5, 7, 1, and 2. These motifs are highly conserved components within the FKBP_C domain, and their relative positions are largely similar across the family.The analysis of conserved domains indicated that, apart from the OsTIG protein, which possesses a Tig domain, the remaining 29 rice OsFKBPs all contain the typical FKBP_C conserved domain characteristic of the FKBP gene family.The domain positions and compositions vary among different subgroups: subgroup I members have an NPL domain at the N-terminus and an FKBP_C domain at the C-terminus; subgroups II and III members have only the FKBP_C domain at the C-terminus; subgroup IV members have only the FKBP_C domain at the N-terminus; subgroup V members possess multiple FKBP_C domains, with some also having a TPR domain at the C-terminus.The gene structure analysis showed that each rice OsFKBP gene contains 4 to 20 coding sequences (CDS), with 27 OsFKBP genes having complete upstream and downstream regulatory regions (UTRs), one OsFKBP gene (OsFKBP16-4) missing the upstream UTR, and two OsFKBP genes (OsFKBP53b, OsFKBP65d) lacking both upstream and downstream UTRs (Figure 5).
Genes 2024, 15, 902 7 of 12 domain at the N-terminus; subgroup V members possess multiple FKBP_C domains, with some also having a TPR domain at the C-terminus.The gene structure analysis showed that each rice OsFKBP gene contains 4 to 20 coding sequences (CDS), with 27 OsFKBP genes having complete upstream and downstream regulatory regions (UTRs), one OsFKBP gene (OsFKBP16-4) missing the upstream UTR, and two OsFKBP genes (OsFKBP53b, OsFKBP65d) lacking both upstream and downstream UTRs (Figure 5).

Analysis of Cis-Acting Elements in FKBP Gene Promoters
Cis-acting elements within the promoter regions of rice OsFKBP genes were analyzed using the PlantCARE online platform, focusing on the 2000 bp upstream of the ATG start codon.The results reveal a substantial presence of light-responsive elements (such as Gbox, Box 4, Sp1, the TCT-motif, and the GT1-motif), plant hormone-responsive elements (including abscisic acid-responsive ABRE, the methyl jasmonate-responsive CGTCA-motif, and the TGACG-motif, as well as the salicylic acid-induced TCA-element), and stressresponsive elements (such as anaerobic induction-ARE, drought response-MBS, and the hypoxia-specific induction-GC-motif) (Figure 6).These findings suggest that OsFKBPs likely play a crucial role in plant responses to photoperiod, hormonal signals, and environmental stressors.
In particular, light-responsive elements are the most prevalent, totaling 293, with 84 Box-4, 43 G-box, and 33 Sp1 elements.Plant hormone-responsive elements are also significant, totaling 280, including 74 ABRE, 71 CGTCA-motif, and 71 TGACG-motif elements.These data indicate that the transcription of OsFKBP genes may be intricately linked to photoperiods and hormonal regulation.Additionally, the genes feature 151 stress-responsive and 71 growth-and development-regulating elements, underscoring their potential regulatory complexity in plant physiology.The prediction of cis-acting elements on gene promoters can provide possible directions and ideas for the study of gene functions.

Analysis of Cis-Acting Elements in FKBP Gene Promoters
Cis-acting elements within the promoter regions of rice OsFKBP genes were analyzed using the PlantCARE online platform, focusing on the 2000 bp upstream of the ATG start codon.The results reveal a substantial presence of light-responsive elements (such as G-box, Box 4, Sp1, the TCT-motif, and the GT1-motif), plant hormone-responsive elements (including abscisic acid-responsive ABRE, the methyl jasmonate-responsive CGTCA-motif, and the TGACG-motif, as well as the salicylic acid-induced TCA-element), and stress-responsive elements (such as anaerobic induction-ARE, drought response-MBS, and the hypoxia-specific induction-GC-motif) (Figure 6).These findings suggest that OsFKBPs likely play a crucial role in plant responses to photoperiod, hormonal signals, and environmental stressors.
In particular, light-responsive elements are the most prevalent, totaling 293, with 84 Box-4, 43 G-box, and 33 Sp1 elements.Plant hormone-responsive elements are also significant, totaling 280, including 74 ABRE, 71 CGTCA-motif, and 71 TGACG-motif elements.These data indicate that the transcription of OsFKBP genes may be intricately linked to photoperiods and hormonal regulation.Additionally, the genes feature 151 stressresponsive and 71 growth-and development-regulating elements, underscoring their potential regulatory complexity in plant physiology.The prediction of cis-acting elements on gene promoters can provide possible directions and ideas for the study of gene functions.

FKBP Gene Response to High-Temperature Stress
To further validate the role the FKBP gene family in rice heat tolerance, the inducibility of selected FKBP genes through high-temperature stress was investigated.In this study, wild-type Nipponbare' rice seedlings were cultivated until day 14, after which they were exposed to high-temperature conditions at 45 °C (16 h of light and 8 h of darkness).The expression patterns of specific members of the FKBP gene family were monitored under these conditions.As depicted in Figure 7, post high-temperature exposure, the expression levels of OsFKBP15-2, OsFKBP15-3, OsFKBP16-3, OsFKBP18, and OsFKBP42b exhibited various degrees of increases.Notably, OsFKBP15-2 and OsFKBP18 demonstrated a significant up-regulation six hours into the heat treatment, which subsequently declined.Conversely, OsFKBP15-3 and OsFKBP42b showed a gradual rise in expression throughout the heat treatment, peaking around 12 h before decreasing.OsFKBP16-3 reached its maximum expression level six hours post-treatment and maintained elevated expression levels from 6 to 24 h compared to its pre-treatment baseline.Collectively, these findings indicate that OsFKBP15-2, OsFKBP15-3, OsFKBP16-3, OsFKBP18, and OsFKBP42b are inducible by high-temperature stress, with OsFKBP15-3 and OsFKBP16-3 exhibiting the most pronounced induction.

FKBP Gene Response to High-Temperature Stress
To further validate the role of the FKBP gene family in rice heat tolerance, the inducibility of selected FKBP genes through high-temperature stress was investigated.In this study, wild-type 'Nipponbare' rice seedlings were cultivated until day 14, after which they were exposed to high-temperature conditions at 45 • C (16 h of light and 8 h of darkness).The expression patterns of specific members of the FKBP gene family were monitored under these conditions.As depicted in Figure 7, post high-temperature exposure, the expression levels of OsFKBP15-2, OsFKBP15-3, OsFKBP16-3, OsFKBP18, and OsFKBP42b exhibited various degrees of increases.Notably, OsFKBP15-2 and OsFKBP18 demonstrated a significant up-regulation six hours into the heat treatment, which subsequently declined.Conversely, OsFKBP15-3 and OsFKBP42b showed a gradual rise in expression throughout the heat treatment, peaking around 12 h before decreasing.OsFKBP16-3 reached its maximum expression level six hours post-treatment and maintained elevated expression levels from 6 to 24 h compared to its pre-treatment baseline.Collectively, these findings indicate that OsFKBP15-2, OsFKBP15-3, OsFKBP16-3, OsFKBP18, and OsFKBP42b are inducible by high-temperature stress, with OsFKBP15-3 and OsFKBP16-3 exhibiting the most pronounced induction.

Analysis of FKBP Gene Responses to Drought Stress
Exposure to drought stress causes significant damage to rice foliage, as documented by Li [30].The cultivation of new drought-resistant varieties will contribute to the development of China's rice industry [31].In this study, wild-type Nipponbare' rice seedlings were cultivated until day 14, followed by exposure to drought conditions using a nutrient solution containing 22% PEG 6000.The expression levels of select members of the FKBP gene family were monitored under these stress conditions.As shown in Figure 8, compared with their levels before drought treatment, OsFKBP15-2, OsFKBP15-3, OsFKBP16-3, OsFKBP18 and OsFKBP42b were significantly induced by drought stress, and OsFKBP15-2, OsFKBP16-3 and OsFKBP18 reached their highest levels at 24 h.The expression levels of OsFKBP15-3 and OsFKBP42b reached the highest levels around 12 h after drought stress, and OsFKBP18 had the highest level of drought induction.These FKBP family members can all be induced to be expresses by drought stress, and OsFKBP15-2, OsFKBP15-3, and OsFKBP18 have the most drastic changes in their expression levels, which may play an important role in the rice response to drought stress.

Discussion
Previous research has demonstrated that the FKBP gene family is integral to both plant growth and development as well as their stress resistance mechanisms [32].Recent advancements have led to the isolation and characterization of numerous FKBPs, revealing critical yet redundant functions within this family.The primary roles of the FKBD structural domain, however, remain elusive [31].Currently, investigations into the FKBP gene family are predominantly conducted in A. thaliana, with significant gaps in our understanding concerning rice.
In this study, bioinformatic tools were employed to identify 30 members of the OsFKBP family from the rice genome, a slight increase from the 29 members reported by Yu, which were based on earlier research by Gollan [32,33].Our findings suggest that this discrepancy may stem from the assembly of newly discovered genes within the rice gene

Analysis of FKBP Gene Responses to Drought Stress
Exposure to drought stress causes significant damage to rice foliage, as documented by Li [30].The cultivation of new drought-resistant varieties will contribute to the development of China's rice industry [31].In this study, wild-type 'Nipponbare' rice seedlings were cultivated until day 14, followed by exposure to drought conditions using a nutrient solution containing 22% PEG 6000.The expression levels of select members of the FKBP gene family were monitored under these stress conditions.As shown in Figure 8, compared with their levels before drought treatment, OsFKBP15-2, OsFKBP15-3, OsFKBP16-3, OsFKBP18 and OsFKBP42b were significantly induced by drought stress, and OsFKBP15-2, OsFKBP16-3 and OsFKBP18 reached their highest levels at 24 h.The expression levels of OsFKBP15-3 and OsFKBP42b reached the highest levels around 12 h after drought stress, and OsFKBP18 had the highest level of drought induction.These FKBP family members can all be induced to be expresses by drought stress, and OsFKBP15-2, OsFKBP15-3, and OsFKBP18 have the most drastic changes in their expression levels, which may play an important role in the rice response to drought stress.

Analysis of FKBP Gene Responses to Drought Stress
Exposure to drought stress causes significant damage to rice foliage, as documented by Li [30].The cultivation of new drought-resistant varieties will contribute to the development of China's rice industry [31].In this study, wild-type Nipponbare' rice seedlings were cultivated until day 14, followed by exposure to drought conditions using a nutrient solution containing 22% PEG 6000.The expression levels of select members of the FKBP gene family were monitored under these stress conditions.As shown in Figure 8, compared with their levels before drought treatment, OsFKBP15-2, OsFKBP15-3, OsFKBP16-3, OsFKBP18 and OsFKBP42b were significantly induced by drought stress, and OsFKBP15-2, OsFKBP16-3 and OsFKBP18 reached their highest levels at 24 h.The expression levels of OsFKBP15-3 and OsFKBP42b reached the highest levels around 12 h after drought stress, and OsFKBP18 had the highest level of drought induction.These FKBP family members can all be induced to be expresses by drought stress, and OsFKBP15-2, OsFKBP15-3, and OsFKBP18 have the most drastic changes in their expression levels, which may play an important role in the rice response to drought stress.

Discussion
Previous research has demonstrated that the FKBP gene family is integral to both plant growth and development as well as their stress resistance mechanisms [32].Recent advancements have led to the isolation and characterization of numerous FKBPs, revealing critical yet redundant functions within this family.The primary roles of the FKBD structural domain, however, remain elusive [31].Currently, investigations into the FKBP gene family are predominantly conducted in A. thaliana, with significant gaps in our understanding concerning rice.
In this study, bioinformatic tools were employed to identify 30 members of the OsFKBP family from the rice genome, a slight increase from the 29 members reported by Yu, which were based on earlier research by Gollan [32,33].Our findings suggest that this discrepancy may stem from the assembly of newly discovered genes within the rice gene

Discussion
Previous research has demonstrated that the FKBP gene family is integral to both plant growth and development as well as their stress resistance mechanisms [32].Recent advancements have led to the isolation and characterization of numerous FKBPs, revealing critical yet redundant functions within this family.The primary roles of the FKBD structural domain, however, remain elusive [31].Currently, investigations into the FKBP gene family are predominantly conducted in A. thaliana, with significant gaps in our understanding concerning rice.
In this study, bioinformatic tools were employed to identify 30 members of the OsFKBP family from the rice genome, a slight increase from the 29 members reported by Yu, which were based on earlier research by Gollan [32,33].Our findings suggest that this discrepancy may stem from the assembly of newly discovered genes within the rice gene database, a conclusion supported by rigorous validation.Moreover, varying strategies in gene identification often result in such discrepancies among studies, as evidenced by Suri and Ge, who identified 71 and 64 members of the wheat FKBP family, respectively [16,34].Following their identification, these OsFKBPs were systematically named from OsTIG, and OsFKBP12 to OsFKBP72, based on their homology with the AtFKBPs from Arabidopsis.Phylogenetic analyses categorized the proteins, except for the distantly related TIG protein, into five distinct subgroups across all chromosomes except chromosome 10.Notably, the distribution of these genes is highly heterogeneous.
Our analysis of conserved sequences and structural domains confirms the high level of conservation observed in both sequence and structure, aligning with findings from earlier studies [4][5][6].The FKBP family has been implicated in numerous stress resistance pathways in plants [16,17].Our promoter analysis identified an abundance of stressresponsive regulatory elements, with a total of 151 elements across the 30 FKBP family members.This is consistent with previous studies [35,36].Then, in order to verify this result, we selected some family members containing regulatory elements of drought stress and high-temperature stress according to the prediction results of the cis-acting elements, and used quantitative RT-PCR to verify whether the expression of these family members was induced by relevant stress (Figures 7 and 8).It was found that the expression levels of these members were indeed up-regulated to varying degrees within 24 h after being subjected to relevant stress, and the expression levels of some members were extremely significant, which suggested that FKBP family members played an important role in the process of the stress resistance of rice.However, there are few studies on the function and mechanism of these OsFKBPs.In addition, we also predicted that the promoters of the FKBP family contain a large number of photo-responsive elements and plant hormoneresponsive elements, which indicates that the transcription of OsFKBP genes may be related to photoperiod and plant hormones.Liu et al. reported that TWD1 (AtFKBP42)-as a described ABCB chaperone-controls stamen development through the activation of AtABCB1,19-mediated auxin transport [37].The continual discovery and characterization of new FKBP family members reveal the complex interplay between these proteins and their molecular chaperones via the FKBD domain.The diverse functionalities of these chaperone proteins contribute to the complexity of FKBP functions.Furthermore, the molecular mechanisms underlying the interactions between FKBPs and their substrates are not yet fully understood and warrant deeper investigation [33].

Conclusions
In this study, 30 FKBP-encoding genes were systematically identified and characterized in rice, which were distributed on all chromosomes and segregated into five distinct subgroups with Arabidopsis except OsTIG and AtTIG.Twenty-nine rice OsFKBPs contain the typical FKBP_C conserved domain characteristic of the FKBP gene family, except for OsTIG.Further analysis of the promoter elements of these rice FKBP genes revealed a high presence of stress-responsive elements.Quantitative PCR assays under drought and heat stress conditions demonstrated that genes OsFKBP15-2, OsFKBP15-3, OsFKBP16-3, OsFKBP18, and OsFKBP42b are inducible by these adverse conditions.These results suggest a significant role for the rice FKBP gene family in stress adaptation.These findings not only enhance our understanding of the FKBP gene family in rice but also provide foundational insights for further detailed studies into their biological roles and mechanisms.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes15070902/s1.Table S1.Basic information on members of the FKBP gene family in rice; Table S2.Primer sequence used in RT-PCR.
Author Contributions: J.Z. conceived and designed the research; F.N. and M.W. performed the experiments and analyzed the data; L.L. and X.M. contributed reagents and materials; J.Z., F.N. and M.W. completed the writing.All authors have read and agreed to the published version of the manuscript.

Figure 1 .
Figure 1.Phylogenetic Tree of FKBPs in rice and Arabidopsis.The phylogenetic tree, constructed using FastTree v2.1.11software, summarized the evolutionary relationships between 53 members of the OsFKBP and AtFKBP families, and named rice OsFKBP family members based on the similarity of the AtFKBP family in Arabidopsis.The tree displays five phylogenetic subfamilies (numbered I to V and marked with different alternating colors).The OsFKBP family in rice was one where internal nodes may not necessarily provide true phylogenetic relationships between different offspring.

Figure 2 .
Figure 2. Localization of OsFKBP genes in rice on chromosomes.Using TBtools, 30 rice OsFKBP genes were mapped onto chromosomes, with each gene's position on the chromosome represented by a line, and the total number of FKBP genes on each chromosome represented by parentheses at the top of each chromosome.This ratio is measured in megabytes (Mb).OsFKBP65d is not anchored to a chromosome.ChrUn, unknown in which chromosome.(1)-(6), the number of identified FKBP distributed on this chromosome.

Figure 2 .
Figure 2. Localization of OsFKBP genes in rice on chromosomes.Using TBtools, 30 rice OsFKBP genes were mapped onto chromosomes, with each gene's position on the chromosome represented by a line, and the total number of FKBP genes on each chromosome represented by parentheses at the top of each chromosome.This ratio is measured in megabytes (Mb).OsFKBP65d is not anchored to a chromosome.ChrUn, unknown in which chromosome.(1)-(6), the number of identified FKBP distributed on this chromosome.

Figure 3 .
Figure 3. Intraspecific collinearity analysis of OsFKBP genes in rice.Using the One Step MCScanX Super Fast component of TBtools v2.041 software, four pairs of collinear genes (connected by red lines) were found within the rice genome.

Figure 4 .
Figure 4. Analysis of collinearity between rice and Arabidopsis FKBP gene species.Use the One Step MCScanX Super Fast component of TBtools v2.041 software to view the collinearity regions in the genomes of rice and Arabidopsis species.Two pairs of collinear genes were found (connected by a red line).
analysis of conserved domains indicated that, apart from the OsTIG protein, which pos sesses a Tig domain, the remaining 29 rice OsFKBPs all contain the typical FKBP_C con served domain characteristic of the FKBP gene family.The domain positions and compo sitions vary among different subgroups: subgroup I members have an NPL domain at the N-terminus and an FKBP_C domain at the C-terminus; subgroups II and III members have only the FKBP_C domain at the C-terminus; subgroup IV members have only the FKBP_C

Figure 3 .
Figure 3. Intraspecific collinearity analysis of OsFKBP genes in rice.Using the One Step MCScanX Super Fast component of TBtools v2.041 software, four pairs of collinear genes (connected by red lines) were found within the rice genome.

Figure 3 .
Figure 3. Intraspecific collinearity analysis of OsFKBP genes in rice.Using the One Step MCScanX Super Fast component of TBtools v2.041 software, four pairs of collinear genes (connected by red lines) were found within the rice genome.

Figure 4 .
Figure 4. Analysis of collinearity between rice and Arabidopsis FKBP gene species.Use the One Step MCScanX Super Fast component of TBtools v2.041 software to view the collinearity regions in the genomes of rice and Arabidopsis species.Two pairs of collinear genes were found (connected by a red line).

Figure 4 .
Figure 4. Analysis of collinearity between rice and Arabidopsis FKBP gene species.Use the One Step MCScanX Super Fast component of TBtools v2.041 software to view the collinearity regions in the genomes of rice and Arabidopsis species.Two pairs of collinear genes were found (connected by a red line).

Figure 5 .
Figure 5. Prediction of the conservative motif (Left), conservative domain (Middle), and the gene structure (Right) of OsFKBPs in rice.

Figure 5 .
Figure 5. Prediction of the conservative motif (Left), conservative domain (Middle), and the gene structure (Right) of OsFKBPs in rice.

Figure 6 .
Figure 6.Prediction of cis elements in the promoter of the OsFKBP gene family in rice.Predictions were made through the analysis of potential cis-acting elements upstream of the ATG 2000 bp starting codon of the OsFKBP gene translation in rice using PlantCARE.Classify and arrange all predicted cis-acting components: Light response (orange), Phytohormone response (green), Stress response (purple), Plant growth and development (yellow).When counting the number of cis-acting elements for each category, the number of cis-acting elements for each category is marked on the right side.

Figure 6 .
Figure 6.Prediction of cis elements in the promoter of the OsFKBP gene family in rice.Predictions were made through the analysis of potential cis-acting elements upstream of the ATG 2000 bp starting codon of the OsFKBP gene translation in rice using PlantCARE.Classify and arrange all predicted cis-acting components: Light response (orange), Phytohormone response (green), Stress response (purple), Plant growth and development (yellow).When counting the number of cis-acting elements for each category, the number of cis-acting elements for each category is marked on the right side.

Figure 7 .
Figure 7. Expression analysis of some members of the OsFKBP genes in response to heat stress.Data are presented as mean ±SD (n = 3), and different letters indicating significant differences among these data at p < 0.05.

Figure 8 .
Figure 8. Expression analysis of some members of the OsFKBP genes in response to drought stress.Data are presented as mean ±SD (n = 3), and different letters indicate significant differences among these data at p < 0.05.

Figure 7 .
Figure 7. Expression analysis of some members of the OsFKBP genes in response to heat stress.Data are presented as mean ±SD (n = 3), and different letters indicating significant differences among these data at p < 0.05.

Genes 2024, 902 9 of 12 Figure 7 .
Figure 7. Expression analysis of some members of the OsFKBP genes in response to heat stress.Data are presented as mean ±SD (n = 3), and different letters indicating significant differences among these data at p < 0.05.

Figure 8 .
Figure 8. Expression analysis of some members of the OsFKBP genes in response to drought stress.Data are presented as mean ±SD (n = 3), and different letters indicate significant differences among these data at p < 0.05.

Figure 8 .
Figure 8. Expression analysis of some members of the OsFKBP genes in response to drought stress.Data are presented as mean ±SD (n = 3), and different letters indicate significant differences among these data at p < 0.05.