Genome-wide characterization and analysis of rice DUF247 gene family

doi:10.21203/rs.3.rs-3866022/v1

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Genome-wide characterization and analysis of rice DUF247 gene family

https://doi.org/10.21203/rs.3.rs-3866022/v1

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published 18 Jun, 2024

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Background

The domain of unknown function 247 (DUF247) proteins are involved in plant development and stress response. Rice is an important worldwide cereal crop, although an increasing number of DUF proteins have been identified, the understanding of DUF proteins is still very limited in rice.

Results

In this study, we identified 69 genes that encode DUF247 proteins in the rice (Oryza sativa) genome by homology searches and domain prediction. All the OsDUF247 proteins were classified into four major groups (I, II, III and IV) by phylogenetic analysis. Remarkably OsDUF247 genes clustered on the chromosomes solely show close phylogenetic relationships, suggesting that gene duplications driven the expansion of the DUF247 gene family in the rice genome. The tissue profile analysis showed that most DUF247 genes expressed at constitutive levels in seedlings, roots, stems, and leaves, except for seven genes (LOC_Os01g21670, LOC_Os03g19700, LOC_Os05g04060, LOC_Os08g26820, LOC_Os08g26840, LOC_Os08g26850 and LOC_Os09g13410) in panicles. These seven genes were induced by various abiotic stress, including cold, drought, heat, hormone treatment, and especially salt, as demonstrated by further experimental analysis. DUF247 proteins contain transmembrane domains located on the membrane, suggesting their significant roles in rice development and adaptation to the environment.

Conclusions

These findings lay the foundation for functional characterizations of DUF247 genes to unravel their exact role in rice cultivars.

DUF247 gene family

gene characterization

phylogenetic analysis

expression pattern

abiotic stress

rice

The domain of unknown function (DUF) is a conserved protein domain whose function remains unknown. DUF proteins usually contain one or more conserved DUF domains and are classified into different families based on the type of DUF domain present [1]. The availability of large amounts of transcriptome and genomic sequence data has led to the identification of numerous DUF proteins. Currently, there are approximately 5889 entries of DUF proteins in the Pfam database [2].

Numerous DUF genes have been studied in different plant species. Previous studies have suggested that DUF proteins are widely involved in various biological processes [3, 4]. Some of them play a key role in plant growth and development. In Arabidopsis, DUF640 and DUF724 have been demonstrated to be involved in seedling development, cell growth, and chloroplast movement [5]. Members of the DUF724 protein family have been found to regulate cell polarity growth in the apical meristem of buds, leaves, or roots [6]. DUF538 domain-containing a pair of proteins was shown to modulate plant growth and trichome development [7–9]. Moreover, TH1/BSG1 which belongs to the DUF640 family is a positive regulator of flower development by controlling inner and outer ridge development [10]. However, OsRMC from the DUF26 gene family negatively regulates root elongation, root numbers, and curliness in rice [11].

In addition to involvement in plant growth and development, DUF proteins have been implicated in stress response as well. The DUF26 has been demonstrated to have a positive regulatory role in plant defense against Ustilago maydis in maize and Pseudomonas syringae in Arabidopsis [12, 13]. On the other hand, AtDUF569 has been shown to have a negative regulatory effect on Arabidopsis resistance to Pseudomonas syringae pv. tomato DC3000 by upregulating the expression of SA-dependent PR genes. The suppression of Arabidopsis RING-DUF1117 E3 ubiquitin ligases, AtRDUF1 and AtRDUF2, reduced tolerance to ABA-mediated drought stress [14]. Overexpression of DUF966 in rice promoted seedling tolerance to high salinity and PEG6000 stress [15]. Expression profiling of ATDUF4228 genes under abiotic stresses such as osmotic, salt, and cold stress suggested that some ATDUF4228 genes may be involved in plant resistance pathways against abiotic stresses [16]. Furthermore, StDDP5 and StDDP7, both of which belong to the DUF221 gene family, exhibited high expression against heat and salt stress, respectively, in potatoes [17].

Although certain DUF gene families have been elucidated, a significant portion of the DUF members remain obscure, particularly in the case of rice. The DUF247 gene family is a large family of genes that encode proteins containing a conserved domain with amino acids ranging from 67 to 711. This gene family is widely present in monocot and dicot plant species, indicating its importance. In Arabidopsis, one member of the DUF247 gene family, DLE1, was found to be a positive regulator of plant defense response [18]. The DUF247 domain-containing LpSDUF247 which protein is considered to be the S-locus male determinant in the gametophytic SI system is highly expressed in pollen and stigma but has low expression in vegetative organs in perennial Ryegrass. [19]. Similarly, OlSS1 and OlSS2, which are homologous to LpSDUF247, are syntenic with OsDUF247 (Os05g0197900) and have been shown to be involved in gametophytic self-compatibility in O. longistaminata [20]. Recently, the DUF247 gene family has been identified as one of the largest and most densely tandemly duplicated gene families in the oak genome [21]. These findings imply that DUF247 genes play pivotal roles in fundamental biological processes in plants, despite the lack of characterization or functional investigation specifically within the context of rice.

In this study, the predicted DUF247-encoding genes were searched in the rice genome and a phylogenetic analysis was conducted. Furthermore, their gene structures, phylogenetic relationships, expression profiles under various abiotic stresses, and protein subcellular localizations were also examined. The studies indicate important functional characterizations of the DUF247 gene family and can be a base and guide for research into the DUF247 gene family in rice.

Evidencing the identification of DUF247 proteins

Sequences of DUF247 proteins from rice, brachypodium, sorghum, maize, and barley were downloaded from phytozome (https://phytozome.jgi.doe.gov). Non-redundant DUF247 proteins were identified using a Hidden Markov Model (HMM). Additionally, the remaining sequences were manually checked for the conserved DUF247 domain using CDD, Pfam, and SMART (http://smart.embl-heigelberg.de/). Furthermore, we performed National Center for Biotechnology Information (NCBI) BLAST analysis to further confirm the DUF247 gene sequences.

Sequence analysis

To better understand the properties of OsDUF247 proteins, we uploaded all protein sequences to ExPASy for calculation of their molecular weights and isoelectric points. Additionally, subcellular localizations were predicted using the Plant-Ploc server (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/).

Phylogenetic analysis

The full-length amino acid sequences of DUF247 proteins were aligned with ClustalW of the software MEGAX using default parameters. The un-rooted phylogenetic tree inferred by the neighbor-joining (NJ) method using MEGAX with the following parameters: Poisson model, partial deletion, and 1000 bootstrap replications [22].

Gene structure and conserved motifs characterization

The software TBtools (Chen et al., 2020) was used to examine the exon-intron structures of rice OsDUF247 genes. Conserved motifs were predicted by the MEME program (http://meme-suite.org/tools/meme).

Chromosomal location, gene distribution and synteny analysis

OsDUF247 genes were mapped to rice chromosomes based on the retrieved annotation file of rice genome. Gene duplication events were analyzed by using Multiple Collinearity Scan toolkit (MCScanX) with the default parameters (Wang et al., 2012). To visualize the syntenic relationship of OsDUF247 genes in the rice genome, the syntenic analysis maps were constructed using the MCScanX.

Plant materials and abiotic stress treatments

Two typical rice varieties, Minghui63 (Indica) and Nipponbare (japonica), were used throughout the experiment. The rice plants were grown in an artificial climate chamber under a photoperiod of 16 hours light/8 hours dark at 28℃. The three-week-old seedlings were transferred to containers with 1/2 MS liquid medium and maintained under the same conditions for one week before abiotic stress treatment, which was carried out as previously described [23]. Tissue samples including root, leaf, stem, panicle, stamen, pollen, and endosperm from different developmental stages were collected separately for RNA extraction and subsequent qRT-PCR analysis. To investigate the expression patterns of selected genes under salt stress, four-week-old seedlings were subjected to a 200 mM NaCl solution for varying durations of 2, 4, 7, 18, and 48 hours.

RNA extraction and qRT-PCR analysis

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and the concentration of each RNA sample was quantified using a NanoDrop ND-1000 spectrophotometer. First-strand cDNA synthesis was performed using the HiScript II 1st Strand cDNA Synthesis kit (Vazyme) following the manufacturer's recommendations. Quantitative real-time PCR (qRT-PCR) was conducted on a Roche Lightcycler 480 II instrument with SYBR Green chemistry. The rice actin gene was used as an internal control. The reaction procedure was as follows: initial denaturation at 95℃ for 30 seconds, followed by 40 cycles of denaturation at 95℃ for 10 seconds, annealing at 60℃ for 30 seconds, and extension at 72℃ for 30 seconds. A final extension step was conducted at 72℃ for 5 minutes. Afterward, the samples were held at 12℃ indefinitely. Each reaction was conducted in triplicate, and the data from qRT-PCR were analyzed using the 2^−ΔCT method. The primer sequences used are listed in Supplementary Table 1.

Expression analysis of OsDUF247 under abiotic stress

The expression pattern of DUF247 genes in the seedling, root, stem, leaf, young panicle, and stamen tissues of the indica cultivar Minghui63 under normal and stress conditions were obtained from the public expression database CREP [24](http://crep.ncpgr.cn/). Additionally, three microarray databases were used to analyze the expression of DUF247 genes under salt stress conditions: GSE6901 for the indica IR64 [25, 26], GSE3503 for the indica IR29 [27], and GSE4438 for indica IR29, MH63, and japonica M103 [28].

The original expression data for OsDUF247 genes under various treatments, including gibberellins (GA), paclobutrazol (PB), salt stress (PRJEB4672), and drought stress (PRJNA272723) were retrieved from the NCBI SRA (Available online: https://www.ncbi.nlm.nih.gov/ ). All the data were analyzed using RNA-seq analysis process, including TopHat, cufflinks, cuffmerge, cuffcompare, cuffquant and using cuffnorm to perform normalization and log transformation. A heat-map was generated using Amazing Heat Map software within the TBtools suite [29] with average linkage hierarchical clustering to show the differential expression levels of OsDUF247 genes across the different treatments.

Identification, characterization and phylogenetic analysis of rice DUF247 proteins among monocot plants

A total of 179 protein sequence entries with a predicted DUF247 domain were found after searching the pubic database through NCBI and Phytozome website with domain number PF03140. To eliminate redundancies, sequences encoding the same proteins and non-representative transcripts were excluded from the analysis. The remaining protein sequences were then screened to ensure the presence of a complete DUF247 domain by HMMER, SMART, CDD, and Pfam. After removing non-DUF247 domain proteins and those with estimated E-values above 1x10^− 10, we identified 69 non-redundant DUF247 genes in the rice genome (MUS7.0). Detailed information regarding the identified DUF247 domains is provided in Supplementary Table 2.

The length of rice DUF247 proteins ranges from 75 (LOC_Os06g08760) to 711 (LOC_Os02g15430) amino acids. The conserved DUF247 domain typically contains about 66–455 amino acids, accounting for the divergent length of the DUF247 genes. The molecular weight of rice DUF247 proteins ranges from 8.68 kDa (LOC_Os06g08760) to 80.03 kDa (LOC_Os02g15430). The DUF247 genes are randomly distributed across 12 chromosomes, except for one gene (PRR), which could not be anchored onto any chromosome. A total of 1,439 pseudogenes were identified with pseudo gene models, and 12% of these genes were expressed within intergenic regions, lacking definable open reading frames. This may explain why the PRR gene could not be anchored onto any chromosome. The predicted isoelectric points of these proteins range from 5.03 (LOC_Os08g42570) to 11.2 (LOC_Os07g47520). Information about DUF247 gene family members including the positions on the chromosomes, the predicted subcellular localizations were presented in Supplementary Table 3.

To investigate the evolutionary relationships among DUF247 members in rice, we used 69 rice DUF247 proteins to construct an unrooted phylogenetic tree using MEGAX with a bootstrap of 1000. The resulting phylogenetic tree (Fig. 1A) showed that the 69 DUF247 proteins were classified into four clades, representing distinct phylogenetic lineages, each supported by a bootstrap value over 80%. Group A and C contained 14 and 15 members, respectively, while groups B and D contained 20 members each. Some genes located on the same chromosomes clustered together in phylogenetic tree, indicating that tandem duplication events may have occurred on the same chromosome.

To investigate the conservation of DUF247 gene family members among monocot species, we used Pfam (PF03140) and HMMER to search the whole genomes of other monocots, including brachypodium, sorghum, maize, and barley. A total of 57, 43, 28, and 59 non-redundant DUF247 genes were detected in their respective genomes (Supplementary Table 4). Phylogenetic analysis revealed that DUF247 proteins from rice, brachypodium, sorghum, maize, and barley could be integrated into six clades, with most of the DUF247 proteins located in clade I (Fig. 1B). The DUF247 family appears to have expanded more rapidly in rice than in other species in a genome size-independent manner.

Gene structure, conserved domain and motif analysis of DUF247 gene family

Given the importance of gene organization in the evolution of gene families (Xu et al., 2012), we determined the gene structures and phases of introns/exons in DUF247 genes by aligning genomic DNA and full-length cDNA sequences. The number of introns varied from 0 to 4 among the DUF247 genes, with 18 (26%) genes lacking introns, 36 (52%) genes containing only one intron, and more than half of the members having more than one intron (Fig. 2A). In plants, the number of introns has been shown to be related to gene expression levels, with more introns generally associated with higher expression levels. However, compact genes may lead to rapid expression when exposed to environmental conditions (Jeffares et al., 2008; Ren et al., 2006).

We identified ten motifs on the conserved domains of DUF247 proteins using MEME program, (Supplementary Table 5). Each protein contained a varying number of conserved motifs, ranging from 2 to 11, and the arrangement of motifs in genes within the same clade was almost identical. Each motif except for motif 10 was found only once in each DUF247 protein sequence. Most members of Clade A shared all ten motifs, with an arranged motif pattern of 3-9-4-2-10-5-6-1-8-7. Some members of Clades B and C shared double "9" motifs. Clade D could be further divided into three sub-groups based on their motif patterns and numbers (Fig. 2B). Sub-group D1 had the fewest number of motifs, while sub-group D2 had an incomplete motif pattern of 3-9-4-2-10-5-6-1-8-7, and sub-group D3 had an almost complete motif pattern, except for motif 10. Furthermore, gene domain analysis revealed a consistent pattern wherein genes within the same group exhibited comparable domain quantities and lengths. Specifically, all members of the DUF247 gene family possessed a solitary domain, while variations in domain length were observed among genes within the same cluster, aligning seamlessly with their corresponding phylogenetic relationships.

As observed through gene domain analysis, members within the same group of DUF247 genes shared similar domain numbers and lengths. All DUF247 gene members were found to have only one domain, and the length of these domains varied among genes within the same cluster, consistent with their phylogenetic relationships (Supplementary Table 2).

In general, closely related DUF247 proteins in adjacent clades or sub-clades of the phylogenetic tree had the same or similar motif structures. The extensive sequence diversity observed in the conserved domain suggests that domain shuffling after genome duplication may have occurred (Morgenstern and Atchley, 1999). However, as the domain range in this gene family is extensive, almost all motifs were included in the domain. This may explain why the motif structure among the four sub-groups was so similar, indicating that no domain shuffling occurred in the structure of the DUF247 protein family.

Chromosome location and syntenic analysis of DUF247 genes

The distribution of DUF247 genes on the chromosomes was uneven, with varying numbers of genes across chromosomes (Fig. 3). The number of DUF247 genes on each chromosome ranged from 1 to 15. Chromosome 8 contained the most DUF247 genes, with a total of 15, while only one gene was located on chromosome 7. These DUF247 genes were observed to be distributed on both the distal and proximal ends of chromosomes.

Gene duplication plays a crucial role in the evolution of plants. Gene family expansion and genomic evolutionary mechanisms mostly depend on gene duplication events. In this study, we identified gene duplication events in the DUF247 gene family. DUF247 gene pairs resulting from segmental and tandem duplications were marked with red lines and red arcs, respectively. As shown in Fig. 3, three tandem duplications were found on chromosomes 8, 11, and 12, accounting for about 10% of the gene family, and were indicated by bending lines. Gene pairs such as LOC_Os08g26220-LOC_Os08g26710 and LOC_Os09g09550-LOC_Os09g12840 have been duplicated several times to form more than one gene pair with the other genes. The duplication on chromosome 12 was too close to resolve their duplication order (detailed duplication information is listed in Supplementary Table 6). Subsequent synteny analysis of the DUF247 gene family revealed an extensive occurrence of over 18 segmental duplication events (Fig. 4). Notably, these duplications transpired not only within genes residing on the same chromosomes but also across different segments of the chromosomes, implying that duplication events likely served as the primary mechanism driving the expansion of the DUF247 gene family in rice. Among the identified duplicated gene pairs, there were a total of 18 segmental duplicates (86%) and 3 tandem duplicates (14%). Remarkably, within the segmental duplicates, 7 of the 18 pairs (39%) belonged to the same phylogenetic clusters. Similarly, 1 out of the 3 tandem duplicates (33%) clustered together phylogenetically. For the remaining gene pairs, close proximity on the phylogenetic tree indicated substantial similarities in their protein domains.

In addition, the synteny analysis of the DUF247 gene family revealed over 18 segmental duplication events (Fig. 4). Duplication not only occurred among genes on the same chromosomes but also among different chromosome segments, indicating that duplication events were the primary mechanism responsible for expanding the DUF247 gene family in rice. A total of 18 pairs of segmental duplicated gene pairs (86%) and 3 pairs of tandem duplicated gene pairs (14%) were identified. For the segmental duplication and tandem duplication, 7 out of 18 and 1 out of 3 pairs were from the same phylogenetic groups, respectively. The other gene pairs were very close in the phylogenetic tree, suggesting that their protein domains had significant similarities.

To gain more insight into the evolutionary constraints acting on the DUF247 gene family, we calculated the Ka/Ks ratio of DUF247 gene pairs. Most segmental and tandem duplicated DUF247 gene pairs had a Ka/Ks ratio < 1 (86%), indicating that the rice DUF247 gene family likely experienced strong purifying selective pressure during evolution. Only 3 the gene pairs had a Ka/Ks ratio > 1, suggesting that positive selection also played a role in the evolution of the DUF247 gene family (Supplementary Table 6).

To further infer the phylogenetic mechanisms of OsDUF247 family, two comparative syntenic maps of japonica rice with two smaller genomic monocot species, including brachypodium and sorghum, were constructed (Fig. 5 and Supplementary Table 7). Twelve and thirteen OsDUF247 genes showed syntenic relationship with those in brachypodium and sorghum, respectively. All genes were found to be associated with its own syntenic gene pairs or syntenic blocks between the rice and other two species. Interestingly, 11 collinear pairs were identified between rice and the other two species, which indicated that these orthologous pairs may already exist before the ancestral divergence. And the collinear pairs were in the same phylogenetic groups, take LOC_Os01g19610.1-Sobic.009G030701.1, LOC_Os02g15430.1-Sobic.004G112400.1 for examples. They all belonged to clade IA (Fig. 1B). The Ka/Ks ratios of these collinear pairs were also calculated, and the majority of orthologous DUF247 gene pairs had Ka/Ks < 1, suggesting that OsDUF247 gene family might have experienced strong purifying selective pressure during evolution. There were also 2 collinear pairs (LOC_Os01g19610.1-Sobic.009G030701.1, LOC_Os05g03972.1-Sobic.009G030701.1) with the Ka/Ks > 1, which suggested that these two pairs might experience a strong positive selection during evolution (the detailed information of collinear gene pairs were listed in Supplementary Table 7).

To gain further insight into the phylogenetic mechanisms of the OsDUF247 family, we constructed two comparative syntenic maps of japonica rice with two smaller genomic monocot species, including brachypodium and sorghum (Fig. 5 and Supplementary Table 7). Twelve and thirteen OsDUF247 genes showed a syntenic relationship with those in brachypodium and sorghum, respectively. All genes were associated with their own syntenic gene pairs or syntenic blocks between rice and the other two species. Interestingly, 11 collinear pairs were identified between rice and the other two species, indicating that these orthologous pairs may have existed before their ancestral divergence. These collinear pairs were in the same phylogenetic groups, such as LOC_Os01g19610.1-Sobic.009G030701.1 and LOC_Os02g15430.1-Sobic.004G112400.1, which both belonged to clade IA (Fig. 1B). The Ka/Ks ratios of these collinear pairs were also calculated, and the majority of the orthologous DUF247 gene pairs had a Ka/Ks ratio < 1, suggesting that the OsDUF247 gene family likely experienced strong purifying selective pressure during evolution. However, there were also two collinear pairs (LOC_Os01g19610.1-Sobic.009G030701.1 and LOC_Os05g03972.1-Sobic.009G030701.1) with a Ka/Ks ratio > 1, which suggested that these two pairs likely experienced strong positive selection during evolution (detailed information of collinear gene pairs is listed in Supplementary Table 7).

The expression pattern of DUF247 genes in rice

To examine the expression patterns of DUF247 genes in various tissues of 63 (MH63) and Nipponbare (Nip) cultivars, we analyzed Affymetrix GeneChip transcriptome data for seedlings, roots, stems, leaves, young panicles, endosperm, and stamen at different developmental stages (Wang et al., 2010). The majority of DUF247 genes had relatively low expression levels across the tested organs and tissues (Fig. 6, Supplementary Table 8). Among the 69 DUF247 genes, LOC_Os08g26220 and LOC_Os02g15500 showed no or very low expression in all tested organs, indicating that they may be pseudogenes or have special temporal and spatial expression patterns not examined in this study. Around 44 genes showed constitutive expression across all detected tissues, while some genes exhibited tissue-specific expression patterns. For example, both LOC_Os01g21650 and LOC_Os01g21670 exhibited high expression in stamen and endosperm, but low expression in leaves. Overall, most DUF247 genes from the same phylogenetic groups shared similar expression patterns. Only 13 genes expressed at higher levels (above 200) in at least one tissue (Supplementary Table 8). We also analyzed three other microarray databases (GSE6901, GSE3053, and GSE4438), which revealed that 6 (46.2%) of genes were present in GeneChips datasets, 3 (23.1%) of genes were common to all three microarrays, and 4 (30.8%) of genes were detected in two GSE databases (Fig. 7a).

To validate the transcriptome profiles of DUF247 genes in different cultivars and tissues, we selected seven genes detected in the chips for qRT-PCR analysis. The results demonstrated that expression levels varied among different tissues (Fig. 7b), and the expression patterns were also different between the indica and japonica rice cultivars (Fig. 7d). For instance, LOC_Os03g19700, LOC_Os05g04060, and LOC_Os08g26820 were highly expressed in the stem and lowly expressed in leaves in both the indica cultivar MH63 and the japonica cultivar Nip, while LOC_Os01g2167 and LOC_Os08g26850 were specifically highly expressed in the stem of MH63 but at a low level in Nip. Notably, all genes were expressed at specifically high levels in panicles in MH63 but at low levels in Nip indicating that some DUF247 genes had low transcriptional activity, although several of them exhibited tissue-specific expression.

Abiotic stress response of DUF247 genes

Our analysis of the microarray database revealed that DUF247 were inducible by salt treatment in the indica cultivar Minghui63 (Fig. 7c). To further investigate their response to abiotic stresses, we searched the SRA database of rice for transcriptome data under salt, drought, gibberellins (GA), and paclobutrazol (PB) treatments (PRJEB4672, PRJNA408068, and PRJNA272723). We found that 26 DUF247 genes were induced by these treatments (Fig. 8, Supplementary Table 9). Interestingly, some genes were significantly induced or repressed by multiple treatments. For example, LOC_Os11g33394 was significantly repressed by drought and slightly repressed by GA, but remained insensitive to salt stress. Most genes were induced by salt stress, including the seven genes detected in the microarray database such as LOC_Os03g19700, LOC_Os08g26220, LOC_Os08g26850, and LOC_Os08g26840.

To gain further insight into the functions of these genes in the evolutionary process, we investigated the response of all seven genes to different abiotic treatments using qRT-PCR (Fig. 7e). Among them, LOC_Os01g21670 and LOC_Os08g26840 were repressed by multiple treatments, while LOC_Os03g19700 was repressed by all treatments except ABA. LOC_Os09g13410 showed repression under cold, drought, heat, and salt treatments, except GA and ABA treatments. Notably, three genes (LOC_Os05g04060, LOC_Os08g26820, and LOC_Os08g26850) detected in all gene chips were significantly induced by salt treatment. Additionally, LOC_Os05g04060 was also induced by heat and ABA treatments, LOC_Os08g26820 was induced by GA treatment, and LOC_Os08g2685 was induced by heat, GA, and ABA treatments.

To further investigate the expression patterns of these three genes, qRT-PCR was performed under 200 mM NaCl treatment at different time points (Fig. 7F). The results showed that compared to the control, all three genes were significantly upregulated after two hours of salt treatment. Subsequently, their expression levels returned to normal within 48 hours of treatment. It suggested that these genes are involved in the response to salt stress and may play a role in the rice plants to saline environments.

The DUF247 gene family represents a large and recently discovered gene family in higher plants. Some members of this gene family have been found to play a crucial role in plant development, particularly in relation to self-incompatibility in grasses [19, 30]. However, the understanding of the overall role of the DUF247 family in development and stress response is still limited. We identified 69 DUF247 genes from the rice genome, which were grouped into four clades based on their evolutionary relationships (Fig. 1). Furthermore, the classification was supported by conserved motif analysis, which revealed that all the identified DUF247 genes shared a common DUF247 domain and transmembrane motif. These findings suggest a potential role for these genes in responding to abiotic and biotic stresses [18].

In monocot grass species, the number of DUF247 genes varies across different species. In rice (genome size: 386 Mb), there are 69 DUF247 genes, while brachypodium (genome size: 272 Mb) has 57, sorghum (genome size: 750 Mb) has 43, maize (genome size: 2,500 Mb) has 28, and barley (genome size: 5,000 Mb) has 59 (Supplementary Table 4). Interestingly, the expansion of the DUF247 gene family in rice appears to be relatively faster compared to other grass species, despite its smaller genome size. It suggests that factors other than genome size are influencing the expansion of this gene family in rice. The clustering of some DUF247 genes on chromosomes suggests the occurrence of duplication events within the gene family. Duplication events drive the distribution and expansion of genes in the genome, and similar observations have been made in other plant gene families, such as the NBS-LRR protein family and F-box protein family [25, 31, 32].

Gene duplication events, including tandem, segmental, and transposition duplications, are essential in genome replication and contribute to the formation of gene families [28]. In the rice DUF247 gene family, three gene pairs evolved from tandem duplication, while 18 segmental duplications, which play a major role in gene family expansion, were identified (Fig. 3, 4). To examine the syntenic relationship between rice and other monocot plants, we analyzed the genomes of two species with smaller genome sizes, brachypodium and sorghum. The results revealed consistent collinearities between rice and these two species, indicating conservation among monocot plants.

Several DUF genes have been implicated in the response to drought and salt stress. GmCBSDUF3 overexpression, containing DUF21 domain, enhanced drought and salt stress tolerance in Arabidopsis [33]. The AhDGR2 gene in Amaranthus hypochondriacus, encoding a DUF642-domain-containing protein, when overexpressed in Arabidopsis, led to increased sensitivity to NaCl treatment [34]. Expression profiling analysis revealed that certain TaDUF966 genes in wheat (Triticum aestivum L.) were induced by salt stress, and TaDUF966-9B was further confirmed to play a role in salt stress through virus-induced gene silencing (VIGS) assay [35]. OsSIDP366, containing DUF1644, positively regulates drought and salt stress responses in rice by regulating PBs/SGs [36]. On the other hand, OsDSR2, encoding a DUF966-domain-containing protein, negatively [15]. Analyses of microarray databases showed that the expression levels of most DUF247 genes were generally low in all investigated tissues or organs, with some genes displaying tissue-specific expression (Fig. 7A), suggesting these genes may represent major functional members of the DUF247 family in response to abiotic stress. Further analysis of the expression patterns of DUF247 genes under various abiotic stresses demonstrated their regulation by multiple stresses, particularly salt stress (Fig. 7E and 7F). Further investigation is needed to gain a deeper understanding of the molecular mechanism underlying the regulation of salt stress by DUF247 genes.

The presence of the DUF247 gene family in the rice genome, as well as their response to various abiotic and biotic stresses, suggests their involvement in stress response and reproductive development. These findings provide valuable insights into the potential functional roles of DUF247 genes. The comprehensive analyses conducted in this study will be beneficial for selecting specific DUF247 genes for further functional characterization and genetic improvement of agronomic traits. Moreover, these findings can contribute to enhancing the environmental resistance of new rice cultivars.

In this study, a total of 179 protein sequence entries with a predicted DUF247 domain were identified and were conducted to estimate evolutionary analysis in rice. Gene structure and conserve motif analysis indicated the conservation and dispersal of DUF247 during evolution. Furthermore, expression profiling and protein interaction predictions suggest that rice DUF247 genes might be involved in plant resistance to environmental stresses. These findings lay the foundation for functional characterizations of DUF247 genes to unravel their exact role in rice cultivars.

DUF247 Domain of unknown function 247

GA Gibberellin

PB Paclobutrazol

HMM Hidden Markov model

Mw Molecular weight

pI Isoelectric point

a. Ethics approval and consent to participate

Not applicable.

b. Consent for publication

Not applicable.

c. Availability of data and materials

All data generated or analysed during this study are included in this published article.

d. Competing interests

“The authors declare that they have no competing interests.

e. Funding

This research was supported by grants from the Science and technology projects of Yunnan Province, (Grant Nos. 202003AD150007), Strategic Leading Science and Technology Program of Chinese Academy of Sciences (Grant Nos. XDA24030301), and Natural Science Foundation of Yunnan, China (Grant Nos. 202101AT070062; 202301AT070351).

f. Authors' contribution

P. Xu, C.K. Lu, F.F. Zhang and J. Yang conceived and supervised this study; F.F. Zhang carried out the experiments, analyzed the data. P. Xu, C.K. Lu, F.F. Zhang and A. Sohail wrote the article. All authors reviewed, edited, and approved the final manuscript.

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No competing interests reported.

SupplementaryTable1.xlsx
Supplementary Table 1: Primers used for quantitative reverse transcriptive PCR.
SupplementaryTable2.xls
Supplementary Table 2: The detailed information of DUF247 domain containing in the 69 candidates.
SupplementaryTable3.xlsx
Supplementary Table 3: The information of characterization of DUF247 members in rice.
SupplementaryTable4.xls
Supplementary Table 4: The distribution of DUF247 genes in the clades in five monocot species.
SupplementaryTable5.xlsx
Supplementary Table 5: Sequences of 10 predicted motifs of DUF247 proteins.
SupplementaryTable6.xlsx
Supplementary Table 6: The detailed information of the DUF247 gene duplication pairs within rice genome.
SupplementaryTable7.xlsx
Supplementary Table 7: One-to-one orthologous relationships between rice and other two plant species.
SupplementaryTable8.xls
Supplementary Table 8: the expression profiles of DUF247 genes in different tissues and organs.
SupplementaryTable9.xlsx
Supplementary Table 9: The detailed information of expression profiles of detected DUF247 genes under normal, salt stress, drought, GA, and paclobutrazol (PB) treatmen

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Genome-wide characterization and analysis of rice DUF247 gene family

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Journal Publication

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Abstract

Background

Results

Conclusions

Figures

Introduction

Materials and methods

Evidencing the identification of DUF247 proteins

Sequence analysis

Phylogenetic analysis

Gene structure and conserved motifs characterization

Chromosomal location, gene distribution and synteny analysis

Plant materials and abiotic stress treatments

RNA extraction and qRT-PCR analysis

Results

Identification, characterization and phylogenetic analysis of rice DUF247 proteins among monocot plants

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1