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Characterization and in silico analysis of the domain unknown function DUF568-containing gene family in rice (Oryza sativa L.)



Domains of unknown function (DUF) proteins are a number of uncharacterized and highly conserved protein families in eukaryotes. In plants, some DUFs have been predicted to play important roles in development and response to abiotic stress. Among them, DUF568-containing protein family is plant-specific and has not been described previously. A basic analysis and expression profiling was performed, and the co-expression and interaction networks were constructed to explore the functions of DUF568 family in rice.


The phylogenetic tree showed that the 8, 9 and 11 DUF568 family members from rice, Arabidopsis and maize were divided into three groups. The evolutionary relationship between DUF568 members in rice and maize was close, while the genes in Arabidopsis were more distantly related. The cis-elements prediction showed that over 82% of the elements upstream of OsDUF568 genes were responsive to light and phytohormones. Gene expression profile prediction and RT-qPCR experiments revealed that OsDUF568 genes were highly expressed in leaves, stems and roots of rice seedling. The expression of some OsDUF568 genes varied in response to plant hormones (abscisic acid, 6-benzylaminopurine) and abiotic stress (drought and chilling). Further analysis of the co-expression and protein–protein interaction networks using gene ontology showed that OsDUF568 − related genes were enriched in cellular transports, metabolism and processes.


In summary, our findings suggest that the OsDUF568 family may be a vital gene family for the development of rice roots, leaves and stems. In addition, the OsDUF568 family may participate in abscisic acid and cytokinin signaling pathways, and may be related to abiotic stress resistance in these vegetative tissues of rice.

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Domains of unknown function (DUFs) are a group of protein families that are highly conserved yet uncharacterized. The first DUFs, DUF1 and DUF2, were identified and renamed the GGDEF domain and EAL domain by Chris Ponting in 1998 [1,2,3]. Since then, the number of known DUF families has increased rapidly owing to the sequencing of genomes in a large number of species. There are 19,632 families, of which, 4795 (24%) (out of 19 632) are DUF families according to the Pfam database version 35.0 [4]. Rice (Oryza sativa L.) is an important cereal crop, and DUFs are predicted to play important roles in its development and responses to abiotic stress [5,6,7,8,9,10].

DUF568 is a conserved domain that is exclusively found in plants. As of January 2023, the Pfam database contained a total of 1,713 sequences belonging to the DUF568-containing gene family (PF04526) across 150 species. An auxin-responsive protein AIR12 (Auxin induced in root cultures), which is a member of the DUF568 family, was reported to interact with other redox partners within the plasma membrane to establish a redox connection between the cytoplasm and the apoplast [11, 12]. In addition, Os03g0194600, also a member of the OsDUF568 family, has previously been reported to be induced by nitrogen starvation in rice roots [13].

This study conducted a comprehensive genomic analysis of the DUF568 gene family in rice, including phylogenetic analysis, subcellular localization prediction, cis-element and expression analysis. Then, the co-expressed genes and interacting proteins of the OsDUF568 family were analyzed to reveal their potential biological functions. Furthermore, the expression of OsDUF568 family in response to phytohormones and abiotic stresses were investigated through experiments. These results would provide valuable insights onto the OsDUF568 family and pave the way for future research into its biological functions.


Identification and phylogenetic analysis of DUF568 family members

The DUF568 domain from Pfam was used to perform HMM (Hidden markov model) searches against the entire protein sequences of rice (Oryza sativa), maize (Zea mays) and Arabidopsis thaliana using local blast (E-value < 10–15). Eight, nine and 11 non-redundant genes were identified in rice, maize and Arabidopsis, respectively (Table 1 and Table S1). The eight DUF568-containing genes from rice were named as OsDUF568.1 to OsDUF568.8 according to their chromosomal order, which were found on chromosomes 3, 8 and 9. The length of OsDUF568 proteins ranged from 193 (OsDUF568.2) to 417 (OsDUF568.1) amino acids, and their predicted theoretical isoelectric points (pI) were concentrated between 8.52 (OsDUF568.8) and 9.74 (OsDUF568.3). The grand average of hydropathicity (GRAVY) scores were estimated to range from 0.08 (OsDUF568.2) to 0.44 (OsDUF568.7), indicating the hydrophilic nature of OsDUF568 proteins. The instability index (II) ranged from 31.05 (OsDUF568.1) to 45.21 (OsDUF568.4). OsDUF568.1, 3, 5 and 7 were predicted to be stable, while OsDUF568.2, 4, 6 and 8 were predicted to be unstable. The predicted subcellular localization showed that OsDUF568. 1, 2, 3 and 4 were located in the apoplast, while OsDUF568. 5, 6, 7 and 8 were targeted to the plasma membrane (Table 1). OsDUF568 proteins were annotated as “DUF568 (and Cytochrom-b561-FRRS1-like) domain-containing protein” according to RAP-DB (The Rice Annotation Project Database). Four members of OsDUF568 family, i.e., OsDUF568.1, 3, 5 and 7, were found to contain a Cytochrom-b561-FRRS1-like domain. In addition, OsDUF568.6 and OsDUF568.8 were recognized as AIR12 [11]. The results suggested that OsDUF568 proteins may have diverse structure and different functions.

Table 1 OsDUF568 gene family and the predicted protein properties

To extend our understanding of OsDUF568 family, neighbor joining tree of DUF568 homologous genes from rice, maize and Arabidopsis thaliana was constructed by bootstrap method (Fig. 1). These DUF568 members were classified into three groups (I, II, III). In three groups, the number of DUF568 members in rice and maize distributed evenly, while eight of nine DUF568 members from Arabidopsis thaliana gathered in Group I, and the remaining one gene (AT3G07390) belonged to Group III. The classification results showed that genetic distance of DUF568 between rice and maize was close, genes in Arabidopsis were far away and more conservative.

Fig. 1
figure 1

Phylogenetic tree showing the evolutionary relationships between DUF568 proteins from rice, Arabidopsis thaliana and maize. The major three phylogenetic clusters were marked as I, II and III based on genetic distance. There were eight, 11 and nine DUF568 proteins from rice (filled circles), maize (unfilled circles) and Arabidopsis thaliana (unfilled square), respectively

Analysis of protein sequences, conserved domains and motifs of OsDUF568 family

Multiple sequence alignment of OsDUF568 proteins was performed using MEGA-X to further understand their homology (Fig. 2A). The conserved amino acid residues (C53, L56, P57, G60, A61, A77, F78, G87, W88, V89, W91, N94, M100, G102, A108, L172, W183, G186, G191, H197) were identified in the range of 40–210, where the DUF568 domain (Purple bar) and DOMON (Dopamine ß-monooxygenase redox domains)-CIL1-like domain (Orange bar) were concentrated. M100 and H197 (Fig. 2A pound signs) play a critical role in coordinating the binding of AIR12 (OsDUF568.6 and 8) and heme [11]. All OsDUF568 proteins contained 20–54 amino-acid signal peptides according to the SignalP website (Fig. 2A), and OsDUF568.1, 3, 5, 6 and 7 possessed TM regions base on the TMHMM website (Table S2). Therefore, it is suggested that OsDUF568.1, 3, 5, 6 and 7 proteins may be secreted proteins, while OsDUF568.2, 4 and 8 are not.

Fig. 2
figure 2

The protein sequences, conserved domains and motifs of OsDUF568. A Multiple sequence alignment of OsDUF568 proteins, showing signal peptides, conserved residues, DUF568 domains, and DOMON-CIL1-like domains. Conservative amino acid residues M100 and H197 were labeled with pound signs (#). Signal peptide sequences were shaded in pink. The purple and orange bars represent the DUF568 domain and the DOMON-CIL1-like domain, respectively. B Conserved domains and motifs of OsDUF568 proteins. Each domain and motif are illustrated with a specific color, and their distribution corresponds to their positions. The length of genes and proteins can be estimated using the scale at the bottom

To investigate the evolutionary relationships of the OsDUF568 family, the conserved domains and motifs were analyzed (Fig. 2B). OsDUF568 proteins all have DUF568 domain, OsDUF568.1, 3, 5 and 7 contained Cytochrom-b561-FRRS1-like domain. Further, nine distinct conserved motifs were identified, motifs 1, 4 and 5 were observed in all OsDUF568s, and motifs 4 and 5 surrounded the region of DUF568 domain in all OsDUF568 proteins, which suggested that motifs 4 and 5 may be essential part of the DUF568 domain. Similarly, motifs 2, 3, 6 and 8 may be related to Cytochrom-b561-FRRS1-like domain. Moreover, motifs 7 and 9 were found in OsDUF568s (OsDUF568.4 excluded) belonged to Group II and III. The structural differences of OsDUF568 proteins suggested that OsDUF568 family may have variant specific functions.

Cis-acting elements of OsDUF568 genes

Analysis of cis-acting elements to a greater detail will facilitate in better understanding the precise control of genes and generate valuable clues for their functional multiplicity [14]. This report manifested potential cis-acting elements in the 2 Kb upstream regions of the OsDUF568 genes from plantCARE website. Thirty-five cis-acting elements were detected totally (Fig. 3A), which formed four main categories as light responsiveness, phytohormone responsiveness, abiotic stresses and plant growth (Fig. 3B).

Fig. 3
figure 3

Identification of cis-acting elements in OsDUF568 genes. A Distribution of cis-acting elements in 2 Kb upstream of each OsDUF568 gene. The different colored boxes indicate distinct elements. B Assessment of different categories and subclasses contained in the OsDUF568 genes. The rose chart on the left shows the proportion of the categories (circle) and subclasses (petal), with the length of each petal proportional to the number of elements. The orange, green, blue and red petals represent light responsiveness, phytohormone responsiveness (including abscisic acid, methyl jasmonate, gibberellin, salicylic acid and auxin), abiotic stress responses (including anaerobic, drought, low-temperature, anoxic, and defense and stress), and plant growth, respectively. The name of the cis-acting elements and their corresponding box colors are sorted from high to low according to their occurrence frequency, accompanied by the subclasses of the cis-acting elements on the right

The four categories contained eleven subdivisions, the largest subdivision was light responsiveness, which contained 45.3% predicted cis-elements, including G-box (Light-responsive element) and Box 4 (Part of a module for light response) as representatives. A series of regulatory elements participating in plant hormone responsiveness ranked second. Cis-acting factors respond to abscisic acid, methyl jasmonate, gibberellin, salicylic acid and auxin were involved. Among them, ABRE (Related to the abscisic acid response) was covered the largest portion, followed by the TGACG-motif and CGTCA-motif (Methyl jasmonate responsive element). In the abiotic stress response category, ARE (Anaerobic induction element) were the most common, followed by those relating to low-temperature responsiveness (LTR) and drought-inducibility (MBS). As for the plant growth regulation category, only two main stress-related cis-acting factors were identified, known as the CAT-box (Referred to meristem expression) and O2-site (Involved in zein metabolism regulation). Intriguingly, all kinds of cis-regulatory elements distributed widely throughout the promoter regions of OsDUF568 genes, revealing that OsDUF568 may have intricate expression patterns and be crucial in the regulation of rice development and stress resistance.

Expression patterns of OsDUF568 genes in different tissues and response to plant hormone & abiotic stresses

To further characterize the potential biological function of OsDUF568 genes (excluding OsDUF568.8), the expression patterns were analyzed in 12 different tissues obtained from RiceENCODE website (Fig. 4). The results showed that the expression levels of OsDUF568 genes varied across different tissues. Specifically, OsDUF568.3 and 5 were highly expressed in most tissues, while OsDUF568.1 was expressed in low levels in most tissues. OsDUF568.4 was barely expressed in nine tissues except for young leaves, nodes I & II, and roots. Importantly, all eight OsDUF568 genes showed high expression levels in these three tissues, indicating that OsDUF568 genes might be involved in the development of leaves, nodes and roots in rice.

Fig. 4
figure 4

Expression patterns of DUF568 genes in different tissues of rice. Log2 transformed min–max normalized gene expression values were used to generate the heat map. The gene expression levels were quantified as fragments per kilobase per million (FPKM) and visualized as a color gradient in the heat map. The color scale bar on the left side of the heat map represents the relative expression level, ranging from 0 to 1.00, where higher values correspond to the higher expression levels

Notably, there were several plant hormone response elements in the upstream cis-acting elements of OsDUF568 genes. To further investigate the possible mechanisms of OsDUF568 genes, this study analyzed the relative expression of OsDUF568 genes in response to six plant hormones (ABA, abscisic acid; GA3, gibberellin A3; IAA, 3-indoleacetic acid; BL, brassinolide; tZ, trans-zeatin; JA, jasmonic acid) using the data from the RiceXPro website (Fig. 5). The relative expression of OsDUF568.4 and 5 were up-regulated after ABA treatment for 3 and 6 h, while OsDUF568.2 and OsDUF568.7 were down-regulated. Most DUF568 genes were insensitive to GA3, IAA and BL treatment, with only OsDUF568.4 showing slight up-regulation after GA3, IAA and BL treatment, and OsDUF568.6 was slightly up-regulated after 3 h of IAA treatment. In addition, OsDUF568.2 and 8 were down-regulated after tZ treatment, while OsDUF568.1, 3, 4, 5, 6 and 7 were up-regulated. Most OsDUF568 genes were down-regulated after JA treatment, except for OsDUF568.3, which showed significant up-regulation. Those results indicated that OsDUF568 genes might regulate relevant hormone signaling pathways. Notably, the expression of OsDUF568.4 changed significantly under the treatment of all six plant hormones, suggesting that OsDUF568.4 may be widely involved in rice hormone signaling pathway.

Fig. 5
figure 5

Relative expression of OsDUF568 genes in response to abscisic acid (ABA), gibberellin A3 (GA3), 3-indoleacetic acid (IAA), brassinolide (BL), trans-zeatin (tZ) and jasmonic acid (JA). The gene expression values were normalized and quantified as Cy5:Cy3 ratios, and visualized as a color gradient in the heat map. The color scale bar on the left represents the relative expression levels, where red indicates up-regulation and blue indicates down-regulation

There were several cis-acting elements related to abiotic stress response in the upstream regions of OsDUF568 genes. The changes in gene expression of OsDUF568 genes in response to various abiotic stresses were analyzed by using publicly available transcriptomic datasets from the Expression Atlas website (Fig. 6).

Fig. 6
figure 6

Differential expression of OsDUF568 family genes in response to anaerobic, drought and chilling. A-B Fold change in the expression of OsDUF568 genes in rice seeds grown under anaerobic conditions (E-GEOD-115371 and E-MEXP-2267). C Differential expression of OsDUF568 genes in susceptible and drought-tolerant rice genotypes under drought conditions (E-GEOD-41647 and E-MTAB-4994). D-F Differential expression of OsDUF568 genes in chilling-sensitive and chilling-tolerant genotypes under chilling stress (E-MTAB-5941, E-GEOD-37940 and E-GEOD-38023). Log2-fold change values were used and visualized as a color gradient in the heat maps

E-GEOD-115371 [15] and E-MEXP-2267 [16] datasets were analyzed (Fig. 6A&B). The E-GEOD-115371 dataset provided molecular profiling of rice seeds grown under anaerobic conditions for 1 h, 3 h, 12 h, 24 h, 2 days, 3 days, and 4 days [15]. As shown in Fig. 6, all OsDUF568 genes showed decreased expression in response to anaerobic stress. Among them, OsDUF568.3 and 4 were strongly down-regulated, reaching the lowest levels at 72 h. The E-MEXP-2267 dataset contained the transcription profiling time course of rice germination under anaerobic conditions, an aerobic to anaerobic switch, and an anaerobic to aerobic switch [16]. OsDUF568.2 and 5 showed decreased expression in response to anaerobic stress, while no OsDUF568 gene showed differential expression in response to the switch between aerobic and anaerobic. Overall, our results showed that the OsDUF568 family were likely to play a role in response to anaerobic stress.

Drought-induced differential gene expression of OsDUF568.2, 3 and 5 from two datasets (E-GEOD-41647 and E-MTAB-4994) were showed (Fig. 6C). E-GEOD-41647 contained data for OsDUF568.2 and 3 from seedlings of susceptible IR20 and drought-tolerant Dagad deshi genotypes [17]. Both OsDUF568.2 and 3 were up-regulated in Dagad deshi, but only OsDUF568.2 in IR24. Moreover, OsDUF568.2 was more up-regulated in Dagad deshi than in IR24, and its expression level increased with drought duration in Dagad deshi. The E-MTAB-4994 dataset contained data for OsDUF568.3 and 5 from the flag leaf at the panicle initiation stage of Nagina 22 (a drought-tolerant genotype) [17]. OsDUF568.3 showed up-regulation while OsDUF568.5 showed down-regulation in response to drought in Nagina 22. The results indicated that OsDUF568 family played a role in response to drought stress.

To understand the role of OsDUF568 family members in chilling/cold tolerance and susceptibility, three datasets (E-MTAB-5941, E-GEOD-37940 and E-GEOD-38023) were analyzed (Fig. 6D, E, F). E-MTAB-5941 contained the data on short- and long-term stress-induced changes in the transcriptome of a chilling-sensitive genotype Thaibonnet and a chilling-tolerant genotype Volano, each subjected to 2 and 10 h chilling treatment at 10 °C [18]. OsDUF568.3 was up-regulated in Thaibonnet and Volano, while OsDUF568.1 and 5 were only up-regulated in Thaibonnet and Volano, respectively. E-GEOD-37940 comprised the transcriptome of the cold tolerant introgression line K354 and its recurrent parent C418 under cold stress [19]. OsDUF568.4 and 7 were up-regulated in K354 and C418, while OsDUF568.3 was up-regulated in C418 only. E-GEOD-38023 contained expression data from a chilling-tolerant Li-Jiang-Xin-Tuan-He-Gu (LTH) japonica landrace variety and a chilling-sensitive IR29 indica cultivar. The plants from both genotypes were subjected to chilling treatment at 4 ℃, and then moved to normal temperature 29 ℃ for 24 h to allow recovery [20]. OsDUF568.2, 3 and 5 were up-regulated in LTH and IR29, while OsDUF568.4 was down-regulated. OsDUF568.7 showed down-regulation in response to normal temperature for recovery. In general, OsDUF568 family genes were differentially regulated in response to chilling between the tolerant and the susceptible rice genotypes.

Co-expression gene networks of OsDUF568 genes

Co-expression network analysis of OsDUF568 genes has the potential to reveal the putative functions of the genes involved in biological processes [21]. The co-expression gene networks of OsDUF568 genes were constructed using the RiceFREND website (Fig. 7A and Table S3). The Weighted Pearson correlation coefficient (PCC) of genes in most networks was around 0.65, while the genes in the network of Os08g0335600 (OsDUF568.4) network had a higher coefficient, suggesting a close functional relationship between the genes in this network. In addition, some genes were co-expressed with both Os03g0194300 (OsDUF568.1) and Os03g0194900 (OsDUF568.3), and most of these genes were related to enzymes such as endoglucanase, caffeic acid 3-O-methyltransferase, and transferase.

Fig. 7
figure 7

Co-expressed gene networks analysis of OsDUF568 genes. A Co-expressed gene networks of DUF568 genes. The red circles represent OsDUF568 genes, the purple circles represent genes from gene ontology (GO) enrichment, and the yellow triangles represent transcription factors. Weighted Pearson correlation coefficients (PCC) are represented by lines, with values close to 0.52 shown as thin black lines, values close to 0.65 shown as blue lines, values close to 0.80 shown as thick red lines. B GO enrichment analysis (Biological process) of OsDUF568s and their co-expressed genes. GO enrichment was found in OsDUF568.4, 5 and 8 networks. C OsDUF568 co-expression genes expression patterns. Data came from RiceXPro, which was performed 75 percentile normalization with log2 transformation and the relative expression value (log2) was obtained by subtracting the median expression value within the data set for each probe

Gene ontology (GO) enrichment analysis was performed to analyze the co-expressed networks of OsDUF568 genes (Fig. 7B). Effective results were obtained for OsDUF568.4, 5 and 8. Network Os09g0501100 (OsDUF568.8) showed significant enrichment with GO terms related to metabolic and stress response, such as ‘oxygen and reactive oxygen species metabolism (GO:0006800)’, ‘response to chemical stimulus (GO:0042221)’, ‘response to oxidative stress (GO:0006979)’ and ‘response to abiotic stimulus (GO:0009628)’ (Fig. 7B). The relative expression of network Os09g0501100 (OsDUF568.8) was high only in roots (Fig. 7C), suggesting that OsDUF568.8 and its co-expressed genes may affect the metabolic and stress response of rice roots. Network Os08g0524200 (OsDUF568.5) mainly consisted of ‘Carboxylic acid transport (GO:0046942)’, ‘Organic acid transport (GO:0015849)’ and ‘Localization (GO:0051179)’, while network Os08g0335600 (OsDUF568.4) contained ‘Lipid transport (GO:0006869)’ and ‘Lipid metabolism (GO:0006629)’ (Fig. 7B). The networks of OsDUF568.4 and 5 were related to transport and highly expressed in leaves, stems and roots (Fig. 7C), indicating that they may participate in the transport of substances during the vegetative growth period of rice.

Remarkably, the co-expressed genes of OsDUF568 networks were highly expressed in roots, stems and leaves (Fig. 7C), which was consistent with the expression patterns of OsDUF568 genes (Fig. 4), suggesting that OsDUF568 and co-expressed genes may play important roles in rice development.

Protein–protein interaction networks analysis of OsDUF568 proteins

The predicted functional partners of OsDUF568s were identified from STRING website, and the protein–protein interaction (PPI) networks were constructed (Fig. 8A and Table S4). The association of most proteins in the OsDUF568 PPI networks were textmined. In addition, there were a number of co-expressed and experimentally determined associated proteins in the networks of OsDUF568.6 and 8. Some proteins were associated with multiple OsDUF568 proteins. Among them, Auxin-repressed protein-like protein ARP1 (OsJ_34778) was associated with OsDUF568.1, 6, and 8, and Pentatricopeptide (PPR) repeat-containing protein-like protein (OS06T0611200-00) was associated with OsDUF568.3, 5, 6 and 7, which indicated the function of the OsDUF568 family may be closely related to these two proteins.

Fig. 8
figure 8

Protein–protein interaction analysis of OsDUF568 proteins. A Interaction network of OsDUF568 proteins. Red circles represent OsDUF568 proteins and white circles represent predicted functional partners of OsDUF568s. Different colored edges represent different protein–protein associations. B Functional enrichment analysis (Biological process) of OsDUF568s and their interacting proteins with FDR < 0.05

According to functional enrichments (Fig. 8B), most networks of OsDUF568 family were related to signal transduction, and proteins in OsDUF568.6 and 8 networks were related to hormone-mediated signaling pathway, especially cytokinin-activated signaling pathway (GO:0009736). Meanwhile, proteins in OsDUF568.1 networks were related to protein deneddylation (GO:0000338) and COP9 signalosome (CSN) assembly (GO:0010387). CSN complex regulates the activity of cullin-RING ligase (CRL) families of E3 ubiquitin ligase complexes, and play critical roles in regulating gene expression, cell proliferation, and cell cycle [22]. The functions of proteins in OsDUF568.7 may include Rab protein signal transduction (GO:0032482) and Cellular localization (GO:0051641), Rab proteins affect cell growth, motility and other biological processes [23]. The results indicated that OsDUF568 family may be involved in material transportation, metabolism and signal transduction in rice.

Expression of OsDUF568 family in response to phytohormones and abiotic stresses

The published data from several public databases above showed that some OsDUF568 family members had higher expression levels in different rice tissues, and were repressed under multiple phytohormones and abiotic stresses, to confirm experimentally, the OsDUF568.2, 3, 4, 6 and 7 expression in rice seedlings subjected to various phytohormones (ABA and 6-BA) and abiotic stresses (drought and cold) treatments were examined. The expression of OsDUF568 genes in leaves, stems and roots of rice seedlings were investigated (Fig. 9A). The OsDUF568.2, 3 and 4 transcript level in stems were lower than leaves and roots, while OsDUF568.6 and 7 were higher. Furthermore, OsDUF568.2, 3, 6 and 7 is highly expressed in the roots. The results suggested the expression of OsDUF568 genes exhibited significant tissue specificity in rice.

Fig. 9
figure 9

Expression profile analysis of OsDUF568 genes in rice seedlings. Relative expression level of OsDUF568 genes in various tissues at rice seedlings (A). Relative expression level of OsDUF568 genes under 6-BA and ABA treatments (B). Relative expression level of OsDUF568 genes under drought and cold treatments (C). Error bars represent ± SD. * and ** indicate the significant difference according to Student’s t-test

The expression of OsDUF568 genes were also regulated by phytohormones. As shown in Fig. 9B, the expression level of OsDUF568.2 decreased after 6-BA treatment, while OsDUF568.3, 4, 6 and 7 reached their highest level after 6-BA treatment for 6 h. Under ABA treatment (Fig. 9B), the expression level of OsDUF568.2 and 7 were suppressed gradually, OsDUF568.3 and 4 were induced to reach highest level after 6 h treatment.

OsDUF568 genes showed significant response to abiotic stresses. OsDUF568.2 expression level reached highest at 3 h after drought treatment, then descend. Expression of OsDUF568.3, and 7 were gradually repressed by drought treatments (Fig. 9C). OsDUF568.6 expression level was induced slightly at the initial time point and suppressed to the lowest level at 12 h. As to cold stress (Fig. 9C), the OsDUF568.2, 4, 6 and 7 expression level were rapidly suppressed. On the contrary, OsDUF568.3 expression level for cold stress was first induced and then suppressed again. These results indicated that OsDUF568 genes were involved in responses to multiple phytohormones and abiotic stresses.


According to the RAP-DB, NCBI (National Center for Biotechnology Information) websites, and results from Preger et al. [11] and us, OsDUF568 family was found to contain DOMON-CIL1-like and Cytochrom-b561-FRRS1-like domains. The DOMON superfamily may be a direct participant in the electron transfer process [24]. Cytochromes b561 (CYB561s) are a family of di-heme transmembrane (TM) proteins that use ascorbate (ASC) as an electron donor and are present in various organs and cell types in plants and animals. The CYB561-core domain is associated with DOMON in ubiquitous CYBDOM proteins, which comprise a novel electron-transfer system potentially involved in oxidative modification of cell-surface proteins. CYB561s and CYBDOMs play important roles in plants such as stress defense, cell wall modifications and cell metabolism [25]. In addition, AIR12s (OsDUF568.6 and 8) were also found to be involved in the establishment of a redox connection between the cytoplasm and the apoplast [11]. Further, the OsDUF568 proteins also contained multiple conserved amino acids (Fig. 2A), with methionine and histidine supporting the binding of OsDUF568 proteins and hemes [11]. All results above showed OsDUF568 family may be involved in stress defense and cell metabolism by mediating electron transport of redox domains in rice.

The upstream regions of OsDUF568 genes were found to contain cis-acting elements that respond to light, phytohormone, and abiotic stresses (Fig. 3), suggesting that these factors may interact to regulate the expression of OsDUF568 genes. The expression patterns of OsDUF568 were investigated in 12 tissues (Fig. 4), and it was found that these genes were highly expressed in rice roots, stems and nodes I and II, particularly in roots, suggesting their importance in rice growth and root development. Furthermore, the relative expression of OsDUF568 genes under six hormone treatments showed that OsDUF568 genes were sensitive to ABA, tZ and JA treatments (Fig. 5). ABA treatment significantly altered the expression of four OsDUF568 genes, while tZ treatment up-regulated most of OsDUF568 genes, and JA treatment down-regulated most. The results indicated that OsDUF568 genes may participate in these hormone pathways in rice.

Rice is adversely affected by abiotic stresses including anaerobic [26, 27], drought [28] and cold [19, 20]. Several reports showed DUFs may be important for rice resistance to abiotic stresses [7,8,9]. All OsDUF568 genes showed decreased expression in response to anaerobic stress (Fig. 6A), indicating that OsDUF568 family were likely to play a role in response to anaerobic stress. Besides, OsDUF568.2 and OsDUF568.3 positively responded to drought stress (Fig. 6B), elucidating their roles in rice adaption to the drought environment. Similarly, OsDUF568.1, 2, 3, and 5 were up-regulated in chilling stress (Fig. 6C), suggesting the four OsDUF568 genes may be important for rice resistance to cold. Considering the common positive response of OsDUF568.2 and OsDUF568.3 under drought and chilling stresses, overexpression of the two genes in rice may be effective methods to engineering plant fitness for drought and cold conditions.

The co-expression (Fig. 7) and PPI (Fig. 8) networks of OsDUF568 genes were constructed, and the possible function of these genes were studied using GO enrichment analysis. The results showed that OsDUF568 genes were widely involved in material transportation, metabolism and signal transduction in rice. Meanwhile, AIR12 (OsDUF568.6 and 8) may be related to hormone-mediated signaling pathway, like cytokinin. OsDUF568.6 was up-regulated while OsDUF568.8 was down-regulated after trans-zeatin treatment, and the two genes showed different expression in response to other phytohormone (Fig. 5). The results indicated the function of AIR12 protein was closely related to phytohormone signal transduction, especially cytokinin-activated signaling pathway.

To understand the potential biological functions of OsDUF568 genes in rice, the RNA transcript levels of OsDUF568.2, 3, 4, 6 and 7 genes in different rice tissues, and treated by phytohormones and abiotic stresses were further investigated in rice seeding (Fig. 9). OsDUF568 genes were generally highly expressed in rice roots, which was consistent with the predicted results (Fig. 4). This indicated that OsDUF568 family may be vital for the development of rice roots. Expression analysis revealed that some OsDUF568 genes were induced or inhibited by different phytohormones treatment. Among them, OsDUF568.4 and 6 were significantly induced after 6-BA treatment. Previous reports showed that OsDUF568.6 would be induced after cytokinin treatment. In addition, cytokinin-inducible type-A response regulator OsRR6 acted as a negative regulator of cytokinin signaling, OsDUF568.4 was highly expressed in rice transgenic lines overexpressing OsRR6 [29]. This suggests that OsDUF568.4 and 6 may be involved in cytokinin signaling pathway. Besides, OsDUF568.3 and 4 were upregulated after both 6-BA and ABA treatment, which suggested OsDUF568.3 and 4 may participate in both abscisic acid and cytokinin signaling pathways. The expression levels of most OsDUF568 genes were decreased under drought and cold stress treatments, while OsDUF568.2 and 3 were significantly induced under drought and cold treatments, respectively. OsDUF568.2 has previously been reported as a gene within a quantitative trait locus (QTL) region for high grain yield under lowland drought [30]. Further research on the biological functions of OsDUF568.2 may be helpful to develop drought resistant versions of popular varieties.

Taken together, these results indicated that OsDUF568 gene family was essential for the development of leaves, stems and roots of rice. The OsDUF568 family may also participate in abscisic acid and cytokinin signaling pathways, and be related to abiotic stress resistance in those vegetative tissues of rice.


This study conducted a comprehensive analysis of the OsDUF568 family. The phylogenetic tree showed a close evolutionary relationship between DUF568 members in rice and maize, while those in Arabidopsis were distantly related. Cis-element prediction displayed that over 82% of the elements upstream of OsDUF568 were responsive to light and phytohormones. Expression patterns revealed that all 7 OsDUF568 genes searched were highly expressed in young leaves, nodes I and II, and roots of rice. Furthermore, the expressions of some OsDUF568 genes were responsive to plant hormones (abscisic acid, trans-Zeatin and jasmonic acid) and abiotic stress (anaerobic, drought and chilling). Further GO analysis of the co-expression and PPI networks revealed that OsDUF568 related genes were enriched in material transportation, metabolism and signal transduction in rice. Finally, RT-qPCR experiments indicated that OsDUF568 family was highly expressed in rice roots, and may participate in signaling pathways involved in phytohormones and abiotic stresses. The findings provide valuable insights into the OsDUF568 family and contribute to the elucidation of their biological functions in the future.


Identification of DUF568 gene family members and phylogenetic analysis

The HMM (Hidden markov model) of the DUF568 (PF04526) domain was obtained from Pfam [31]. The HMM was compared with the whole protein sequences of rice, Arabidopsis thaliana and maize obtained from the NCBI [32] using HMMER ver. 3.0 (E-value < 10–15) [33]. The MSU and RAP loci of DUF568 genes in rice were obtained from the China Rice Data Center [34]. Exons and chromosome locations were obtained from the NCBI [32], and description were obtained from RAP-DB (The Rice Annotation Project Database) [35]. Protein physicochemical properties were analyzed using the ProtParam [36], and subcellular localization was predicted using the PSORT [37].

The full length DUF568 protein sequences of rice, Arabidopsis and maize were compared in MEGA-X. Multiple sequence alignment was performed using the MUSCLE aligner with all other parameters set to the default settings. The neighbor joining tree was constructed using the bootstrap method with 1000 repetitions.

Comparison of protein sequence, gene structure, conserved domains and motifs

The amino acid sequences of OsDUF568 proteins in rice were compared using the MUSCLE method in MEGA-X and visualized using Jalview. The signal peptides were identified using the SignalP [38], and the transmembrane regions were analyzed using the TMHMM [39]. The conserved motifs of OsDUF568 proteins were predicted using the MEME [40], domains were obtained from Pfam, and both were visualized using TBtools [41].

Cis-acting elements

The genome annotation for rice was obtained from NCBI [42]. The cis-acting elements located within 2 Kb upstream of the OsDUF568 genes were extracted using the plantCARE website [43] and visualized using TBtools [41]. The rose chart was created using Microsoft Office PowerPoint 2019.

Expression patterns

The expression patterns of OsDUF568 genes in 12 different tissues (including flower buds, flowers, panicles, milk grains, mature seeds, endosperm, young leaves, mature leaves, lamina joints of flag leaf, stem, nodes I and II, and roots) were obtained from the RiceENCODE [44]. The expression patterns in response to plant hormones (including ABA, GA3, IAA, BL, tZ and JA) were obtained from the RiceXPro [45]. Gene expression data for OsDUF568 genes under abiotic stress conditions were extracted from several datasets available at the EMBL-EBI Expression Atlas website [17] including E-GEOD-115371, E-MEXP-2267, E-GEOD-41647, E-MTAB-4994, E-MTAB-5941, E-GEOD-37940 and E-GEOD-38023. Log2-fold change values were used and visualized as a color gradient in the heat maps. All data were visualized using TBtools [41].

Co-expressed genes and PPI networks

The co-expressed genes of OsDUF568 genes were searched using RiceFREND [46], Gene ontology (GO) terms were obtained from the GO [47] with P < 0.05 and FDR (False discovery rate) < 0.05. The expression patterns of co-expressed networks in different rice tissues were obtained from the RiceXPro website [48], and visualized using R. The PPI networks and functional enrichments results of OsDUF568 proteins were obtained from STRING [49]. Both networks were drawn by Cytoscape ver. 3.9.0 [50].

Phytohormone treatments and abiotic stress treatments

The rice cultivars japonica Nipponbare was used for all RT-qPCR analysis. The rice seeds were soaked in 75% alcohol for 1 min, 20% Sodium hypochlorite for 15 min for surface disinfection, and clean the seeds 10 times with water. The seeds were soaked in a fresh water at 28 ℃ for 24 h and germinate for 24 h at 37 °C. Germinated seedlings were transferred to a IRRI (International rice research institute) hydroponic system. Plants were grown in a growth-chamber at 30 °C / 25 °C in a 16-h-light / 8-h-dark cycle and with 75% humidity.

7-day-old seedlings were used to examine the expression patterns of OsDUF568.2, 3, 4, 6 and 7. The leaves, stems and roots were sampled from seedlings without any treatment. For drought stress, the roots of seedlings were immersed in 15% PEG-6000 for drought stress. For cold stress, seedlings were transferred to a growth chamber at 4 ℃. The various treated roots were sampled at 0, 1, 3, 6, 12, 24 and 48 h after the abiotic stresses. Phytohormone treatments were performed by adding into the hydroponic system with 50 μM abscisic acid (ABA) and 1 μM 6-benzylaminopurine (6-BA) respectively, and then the roots were sampled at 0, 1, 3 and 6 h after phytohormone treatments. These collected samples were immediately frozen in liquid nitrogen and stored at -80 ℃. Three replications were performed.

Isolation of RNA, real-time quantitative PCR and expression analysis

Total RNA was isolated from the collected samples using the RNApure Plant kit (CWBIO, Nanjing, China). Using ToloScript All in one RT EasyMix for qPCR (TOLOBIO, Nanjing, China) to remove residual genomic DNA and synthesize first-strand cDNA. RT-qPCR was performed with 2 × Q3 SYBR qPCR Master mix (TOLOBIO, Nanjing, China) in a final reaction volume of 10 μL using an Bio-Rad CFX Connect Real-Time PCR Instrument (Bio-Rad, Bio Rad, Hercules, USA). OsActin (Gene ID: 4333919) served as internal controls. Expression levels are depicted as cycle threshold (Ct) value of the candidate gene relative to the Ct value of the housekeeping gene. Data were analyzed with the Bio-Rad CFX Manager software and visualized using R. All gene-specific primers are listed in Table S5.

Availability of data and materials

All data generated or analyzed during this study are included in this article and its additional files. All data of the co-expressed genes and potential interacting proteins of the OsDUF568 family obtained based on the open databases, and the results of the GO enrichment analysis were available in Table S3 and S4.



Abscisic acid


Auxin induced in root cultures


Anaerobic induction induction element






Cytochromes b561


Domains of unknown function

GA3 :

Gibberellin A3


Gene ontology


Grand average of hydropathicity


3-Indoleacetic acid


Instability index


Jasmonic acid




Lipid transfer protein


Low-temperature responsiveness




National center for biotechnology information


Isoelectric points


Protein-protein interaction


Quantitative trait locus


The rice annotation project database


Response regulator


Real-time quantitative PCR






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The authors sincerely thank Jiayin Pang from The University of Western Australia (Perth, Australia) for providing suggestions and polishing of manuscript.


This work was financially supported by the National Natural Science Foundation of China (31471574), The open funds of the Key Laboratory of Plant Functional Genomics of the Ministry of Education (NO. ML202104), the Open Project Program of Jiangsu Province Engineering Research Center of Knowledge Management and Intelligent Service, Yangzhou University (KMIS202207) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX22_3469).

Author information

Authors and Affiliations



The study was conceived by KC and YW under the supervision of BL; KC conducted analysis and drafted the manuscript; YW, XN and YZ performed the expression and genetic analyses; TT, YC and QS provided scientific feedback. CY and BL reviewed and edited the manuscript. All authors have read and approved the final manuscript.

Corresponding author

Correspondence to Bing Lü.

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The plant materials of the cultivars used in this study were commercially available. Plant materials used in the analysis are maintained in accordance with the institutional guidelines of Yangzhou University, China. This article did not contain any studies with human participants or animals and did not involve any endangered or protected species.

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Supplementary Information

Additional file 1: Table S1.

DUF568 family in Arabidopsis and maize.

Additional file 2: Table S2.

Trans-membrane regions prediction of OsDUF568 family.

Additional file 3: Table S3.

Co-expressed genes of the OsDUF568 family and gene-ontology enrichment analysis.

Additional file 4: Table S4.

PPI networks of the OsDUF568 family and functional enrichment analysis.

Additional file 5: Table S5.

Gene-specific primers used for RT-qPCR experiments.

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Chen, K., Wang, Y., Nong, X. et al. Characterization and in silico analysis of the domain unknown function DUF568-containing gene family in rice (Oryza sativa L.). BMC Genomics 24, 544 (2023).

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