Next Article in Journal
Melatonin Mediates Cardiac Tissue Damage under Septic Conditions Induced by Lipopolysaccharide
Next Article in Special Issue
Single-Base Methylome Analysis of Sweet Cherry (Prunus avium L.) on Dwarfing Rootstocks Reveals Epigenomic Differences Associated with Scion Dwarfing Conferred by Grafting
Previous Article in Journal
Alteration in sB7-H4 Serum Levels and Placental Biomarker Expression after Therapeutic Plasma Exchange in Early-Onset Preeclampsia Patients
Previous Article in Special Issue
Integration of Transcriptome and Metabolome Reveals Wax Serves a Key Role in Preventing Leaf Water Loss in Goji (Lycium barbarum)
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 25
Issue 20
10.3390/ijms252011087
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Comprehensive Analysis of BrDUF506 Genes Across the Brassica rapa Genome Uncovers Potential Functions in Sexual Reproduction and Abiotic Stress Tolerance

by
Guangqi Zhu
1,2,†,
Jingxuan Wang
3,†,
Shuang He
2,
Kexin Liang
2,
Renyi Zhang
2,
Jiabao Huang
2,
Xueqin Yang
1,* and
Xiaojing Zhang
1,*
1
College of Biology, Hunan University, Changsha 410082, China
2
Institute of Vegetable Science, Zhejiang University, Hangzhou 310058, China
3
College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an 271000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2024, 25(20), 11087; https://doi.org/10.3390/ijms252011087
Submission received: 31 August 2024 / Revised: 8 October 2024 / Accepted: 12 October 2024 / Published: 15 October 2024
(This article belongs to the Collection Advances in Molecular Plant Sciences)
Browse Figures
Versions Notes

Abstract

:
The Domain of Unknown Function 506 (DUF506) belongs to the PD-(D/E) XK nuclease superfamily and has been reported to play critical roles in growth and development as well as responses to abiotic stresses. However, the function of DUF506 genes in Brassica rapa (B. rapa) remains unclear. In this study, a total of 18 BrDUF506 genes were identified and randomly distributed across eight chromosomes, categorized into four subfamilies. Analyzing their promoter sequences has uncovered various stress-responsive elements, such as those for drought, methyl jasmonate (MeJA), and abscisic acid (ABA). Bra000098 and Bra017099 exhibit significantly enhanced expression in response to heat and drought stress. Protein interaction predictions indicate that Bra000098 homolog, At2g38820, is interacting with ERF012 and PUB48 and is involved in abiotic stress regulation. Furthermore, gene expression profiling has identified Bra026262 with a high expression level in flowers and significantly decreased in female sterile mutants. Protein interaction prediction further revealed that its homolog, At4g32480, interacts with MYB and AGL proteins, suggesting the potential roles in female gametophyte development. The current study enhances our understanding of the functional roles of BrDUF506s, providing significant insights that are valuable in investigating sexual reproduction and abiotic stress responses in B. rapa.

1. Introduction

The Domain of Unknown Function (DUF) is a category of gene families that have conserved domains whose functions remain undetermined [1]. While many DUF families remain mysteries, research has been conducted on some. Proteins with DUF domains are involved in a range of biological processes, including modifying cell walls, producing lignin, and influencing microtubule dynamics [2,3,4]. This suggests a broad role for these proteins in various cellular functions.
Studies suggest that DUF genes are involved in various reactions to abiotic stress. The OsDUF568 gene family, for instance, is essential for the growth of rice roots, leaves, and stems and is likely engaged in signaling pathways associated with abscisic acid and cytokinin, thereby enhancing the resistance of rice vegetative tissues to abiotic stress [5]. OsMIZ1, a member of the OsDUF617 family, has been implicated in drought and salt stress tolerance, as well as in developmental and stress response processes in rice [6]. ESK1, a representative of the AtDUF231 family, has been identified as a negative regulator in the context of freeze tolerance [7]. Furthermore, DUF genes also play a pivotal role in sexual reproduction. A study has identified causal genes for postzygotic reproductive isolation in rice hybrids, demonstrating that mutations in DUF1668-containing genes contribute to hybrid sterility and potentially drive speciation [8]. Another study has revealed that in seed plants such as Arabidopsis and tobacco, the HAP2/GCS1 fusogen interacts with sperm AtDUF679 membrane proteins DMP8 and DMP9, which are crucial for its translocation to the sperm plasma membrane in response to egg cell signals, thereby ensuring successful fertilization through a conserved mechanism [9].
The DUF506 family is classified within the PD-(D/E)XK nuclease superfamily [10]. Research on the DUF506 family has been conducted in Arabidopsis and Oryza sativa (O. sativa). AtRXR3, a phosphorus stress-inducible AtDUF506 protein in plants, negatively regulates root hair growth [11]. The Arabidopsis AtDUF506 family is widely present in photosynthetic organisms and shows potential roles in coping with environmental stresses and nutrient shortages [12]. In rice, the OsDUF560 family was actively engaged in the response to abscisic acid (ABA) and jasmonic acid (JA), exhibiting distinct expression profiles under drought and cold stress. This knowledge could be advantageous for future crop breeding efforts [13]. However, as of now, the BrDUF506 genes in B. rapa have not undergone genome-wide identification, and their functions remain unexplored.
B. rapa, a vegetable extensively consumed within the Brassicaceae family, encounters dysgenesis and a range of abiotic stresses that undermine its productivity [14]. In light of the escalating global demand for vegetables, there is an imperative to identify genes capable of mitigating these issues and enhancing agricultural output. This study identified the BrDUF506 genes at the genomic level and analyzed their physical and chemical properties, structure, gene, protein, and expression profiles. These findings offer critical insights for future research on BrDUF506 genes in B. rapa.

2. Results

2.1. Identification and Physicochemical Characterization of DUF506 Gene Family in B. rapa

By comparing the homologous genes of the AtDUF506 gene family with the B. rapa genome database, we found 18 BrDUF506 genes and categorized them into four subfamilies. The genes were named based on their locations within the chromosomes. The physicochemical characteristics were summarized in Table 1. The BrDUF506 genes are spread out over 8 of the 10 chromosomes. Bioinformatics analysis showed that the predicted isoelectric point (pI) ranged from 5.34 (Bra033896) to 10.78 (Bra040373), the protein lengths varied from 192 aa (Bra040373) to 367 aa (Bra033896), and MW ranged from 22 kDa (Bra040373) to 41 kDa (Bra033896). The length of the protein is linked to its molecular weight in a positive way. The majority of BrDUF506 proteins were predicted to be localized to the nucleus, with a few likelihoods in the cytosol and chloroplast (Table 1 and Supplementary Figure S1). Bioinformatics analysis suggests that BrDUF506 proteins might have various functions, depending on how they are distributed among the organelles.

2.2. Chromosomal Distribution and Phylogenetic Relationships of BrDUF506s

In total, eighteen BrDUF506 genes have been identified in the B. rapa genome and are distributed non-uniformly across eight chromosomes, with chromosome 3 harboring the most BrDUF506 genes, followed by chromosome 7, having four BrDUF506 genes. While there are no BrDUF506 genes distributed on chromosomes 2 and 9 (Figure 1A). In order to elucidate the evolutionary relationships among BrDUF506 genes, we conducted a maximum likelihood (ML) phylogenetic tree of DUF506 genes between B. rapa and A. thaliana (Figure 1B). The 31 DUF506s were clustered into four groups (I, II, IIIa, and IIIb); Group I and Group IIIb, comprising 9 members, were the largest groups. Group IIIa contained 7 DUF506 members: 4 BrDUF506s and 3 AtDUF506s, and Group II was the smallest group, having 6 members (Figure 1B). Overall, the classification of BrDUF506 members is largely concordant with the A. thaliana phylogenetic tree, suggesting a close relationship between B. rapa and A. thaliana.

2.3. Collinearity Analysis of BrDUF506 Members

Gene duplications are significant for gene family evolution [15]. The collinearity analysis result showed ten segmental duplications, with Bra024209 harboring the most duplications: Bra024209-Bra031136, Bra024029-Bra026262, and Bra024209-Bra036492, followed by Bra013223, Bra036492, Bra036877, and Bra033896 having two duplications, respectively (Figure 2A). This indicates that segmental duplication is a pivotal mechanism in the evolutionary expansion of the BrDUF506 gene family.
In addition, to enhance our understanding of the origin and evolutionary history of DUF506 genes, we also identified the duplicated events of DUF506s in B. rapa, the dicot model plant (A. thaliana), and the monocot species (O. sativa) (Figure 2B). Twenty-two pairs of collinear genes were identified between BrDUF506 and AtDUF506, whereas only four pairs were found between BrDUF506 and OsDUF506 (Figure 2B). The findings reveal BrDUF506s have a greater degree of collinearity with AtDUF506s in comparison to OsDUF506s, indicating a significant relationship and functional similarity between BrDUF506s and AtDUF506s.

2.4. Characterization and Conserved Motifs of BrDUF506s

A novel phylogenetic tree of BrDUF506 proteins was constructed through the analysis of full-length protein sequence alignments and categorized into four distinct groups (I, II, IIIa, and IIIb) (Figure 3A). It was noted that the BrDUF506 protein number exhibited a relatively even distribution across each of these groups. An analysis of the exon-intron configuration of the BrDUF506 genes was performed, and it exhibits significant structural diversity, with Bra029641, Bra040373, and Bra013223 in group I only having a single exon, while the other group I genes (Bra016797 and Bra019736) harboring two exons (Figure 3B). Those BrDUF506s in groups II, IIIa, and IIIb possessed two or three CDS regions (Figure 3B). It suggested that members of different subfamilies might have distinct functions. Furthermore, eighteen conserved motifs of BrDUF506s were identified. These motifs vary in number and placement across BrDUF506s, but all BrDUF506 members shared motifs 1, 3, 2, and 6 (Figure 3C). The conserved motifs in Group I exhibit the most significant differences, indicating potential functional diversification. Group II, IIIa, and IIIb each shared the same motifs, indicating the same subfamily may have the same biological function. In addition, motif 4 is only found in group II, and motif 7 is a group IIIb special (Figure 3C); this kind of structural similarity might hint at their specific functions in B. rapa.

2.5. Analysis of Cis-Acting Elements of BrDUF506s Promoter

Promoters act as essential “switches” for genes, triggering gene transcription and governing gene activity [16]. For further analysis of the prospective functions of BrDUF506s, we used PlantCARE to conduct an analysis of the 2 kb upstream region of the BrDUF506s coding region for cis-acting element prediction (Figure 4). Cis-acting elements were classified into three groups based on their function: growth and development, phytohormone response, and stress response. Figure 4 illustrates the quantity and arrangement of these elements for each gene. In the first category (phytohormone response), most genes had various phytohormone response elements, with 100% containing MeJA-responsive elements, 72.2% harboring auxin response elements, 55.5% having ABA-responsive elements, and the fewest containing GA-responsive elements, only 38.8% (Figure 4). Furthermore, the majority of BrDUF506 genes possess estrogen-responsive elements, indicating their potential role in sexual reproduction (Figure 4).
In the stress response group, all BrDUF506 genes contain acid response elements, and most genes harbor three or four types of stress response cis-acting elements, indicating that certain genes are key players in responding to stress. (Figure 4). The group IIIb gene Bra000098 was found to encompass four different types of elements and boasts the highest number of elements. Similarly, other group IIIb genes Bra017099, Bra036877, Bra033896, and Bra001897 each possess a variety of element types, with more than ten elements in total (Figure 4). Consequently, it is highly probable that the BrDUF506 genes in Group IIIb play a significant role in tolerating abiotic stress.

2.6. Analysis of Abiotic Stress Transcript Levels of BrDUF506s

To evaluate the role of the BrDUF506 gene family in stress response mechanisms, we conducted an analysis of their genes transcriptome expression profiles subsequent to stress induction. The genes Bra000098, Bra017099, Bra031136, and Bra029641 demonstrated significantly elevated expression levels in response to heat stress (Figure 5A; Supplementary Table S1). Furthermore, Bra000098 and Bra017099 also showed a notable increase in expression under drought stress conditions (Figure 5B). Additionally, a comparable trend is evident in AtDUF506s (Supplementary Figure S2). These results suggest that the BrDUF506 gene family contributes to abiotic stress tolerance, with Bra000098 and Bra017099 being particularly noteworthy.

2.7. Expression Pattern of BrDUF506 Members in Different Tissues

An analysis of expression values in different tissues was performed to further investigate the expression specificities of the BrDUF506s. The data indicate that while the expression of these genes is generally low across most tissues, some genes see a significant increase in expression in specific tissues, suggesting they may have tissue-specific functions. Our analysis revealed that Bra031136 was prominently expressed in callus, indicating that it potentially contributes to plant regeneration (Figure 6; Supplementary Table S2). Similarly, Bra029641 displayed high expression in silique, but the homologous Bra040373 does not have a similar expression pattern, presumably because they are evolutionarily different (Figure 6). Furthermore, AT3G07350 was more related to Bra029641 than Bra040373, thus Bra029641 and Bra040373 may have evolved different protein structures with diversity-conserved motifs that lead to different functions. Additionally, Bra000140 was found to be highly expressed across various plant parts, including the root, leaf, and stem, possibly because its unique protein structure may be involved in a variety of biological functions (Figure 6). In flowers, both Bra026262 and Bra024209 showed high expression levels, suggesting group II genes are likely to be involved in sexual reproduction (Figure 6).

2.8. Sexual Reproduction-Related Expression Profiling of BrDUF506s

In order to determine whether BrDUF506s may be involved in sexual reproduction, we analyzed sexual reproduction-related expression profiling of BrDUF506s from the Chinese cabbage male sterile mutant (msm) and female sterile mutant (fsm) (Figure 7; Supplementary Table S3). Bra001897 and Bra033896 showed upregulation in msm, while Bra017099 manifested an obvious down-regulated expression value in male sterile mutants (Figure 7A), indicating that these three genes may not only be involved in abiotic stress but also in sexual reproduction. Moreover, we found a distinct decline of Bra026262 in fsm (Figure 7B), suggesting that it may regulate pistil growth and development.

2.9. Prediction of Protein-Protein Interaction (PPI) Network

Proteins are fundamental in performing diverse cellular functions, and they physically interact with several types of molecules, including lipids, nucleic acids, and metabolites [17,18]. Considering the tight evolutionary bond between B. rapa and A. thaliana, we are making educated guesses about the biological roles of similar genes in B. rapa by analyzing how the DUF506 genes in A. thaliana interact with proteins. The PPI network analysis utilized a public STRING database to conduct the predicted protein interaction map for three vital BrDUF506 genes (Bra017099, Bra000098, and Bra026262) that are related to abiotic stress and sexual reproduction (Figure 8; Supplementary Table S4). We have identified some functional genes, including RZPF34 [19], ERF012 [20], and PUB48 [21,22] (Figure 8A), which are known to be involved in the ABA and ethylene response mechanisms. These genes are crucial in enhancing plant tolerance to abiotic stress. Interesting, we have also discovered genes DUF1/6 (DUF724 domain-containing protein 1/6) (Figure 8A). Research on the DUF724 domain has shown its involvement in floral development [23]. We also identified genes MYB59 [24,25], PDC1 [26], and AGL56 [27] (Figure 8B), which are involved in plant growth and stress responses. This finding provides additional evidence of the functional diversity within the BrDUF506 gene family.

3. Discussion

Consequent to anthropogenic activities and the phenomenon of climate change, there is an escalating incidence of multiple abiotic stresses on plant species. Such as severe temperature fluctuations, prolonged drought, excessive flooding, heightened light intensity, and modified salinity levels [28]. Furthermore, the issue of dysgenesis in plants is critically influencing the establishment and diversification of germplasm resources [29]. Previous research has underscored the vital importance of DUF506s in plant growth, development, reproduction, and coping with abiotic stress [11,12,13]. Nevertheless, the functional and family analysis of DUF506 genes in B. rapa has not been previously investigated. In the present study, we utilized bioinformatics methodologies to examine the BrDUF506 gene family features, gene expression profiles, and regulatory mechanisms. This research is anticipated to facilitate subsequent functional inquiries into the BrDUF506 gene family.
This study identified 18 members of the DUF506 gene family in B. rapa. Classification of these genes into four clusters was based on their phylogenetic relationships and sequence similarities. Collinearity analysis suggests that BrDUF506s is more closely related to A. thaliana than O. sativa.
Motif analysis has indicated the potential involvement of Bra000098 and Bra017099 in the regulation of responses to abiotic stress. Transcriptome data analysis has revealed a significant up-regulation of these genes in response to heat stress and drought conditions. Analysis of protein-protein interaction networks has revealed potential interactions among At2g38820 (homologs: Bra000098 and Bra017099) and other cellular factors. Notably, an interacting protein, GRF11, is recognized as a critical regulatory component in the nitric oxide pathway, mediating responses to iron deficiency in A. thaliana [30]. At2g38820 interacts with CGLD27/At5g67370, which has been identified as important for the growth of A. thaliana under iron-limited conditions [31]. Studies have demonstrated that extracellular iron not only serves as an iron reservoir but also plays a significant role in modulating plant responses to environmental changes through targeted deposition [32]. Our research identified other interacting proteins, ERF012, an ethylene response factor, and PUB48, a member of the U-box protein family. ERF012 is involved in regulating stress responses through the modulation of auxin accumulation and ethylene biosynthesis [20]. Overexpression of AtPUB48 has been shown to induce the expression of heat-related genes, including HSP101, HSP70, HSP25.3, HSFA2, and ZAT12, thereby enhancing plant resistance to heat stress during seed germination and seedling growth [22]. Moreover, AtPUB46 and AtPUB48 contribute to the regulation of plant sensitivity to drought and the inhibition of seed germination by abscisic acid (ABA), highlighting their significant roles in stress response mechanisms [21]. The above indicates that BrDUF506s may play a role in the regulation of abiotic stress responses through the mediation of hormones such as abscisic acid (ABA) and ethylene. Identify the potential pathways through which the BrDUF506 gene family manages abiotic stress. Under conditions of heat or drought stress, Bra000098 and Bra017099 interact with ERF012 and PUB48, which in turn control genes that respond to stress, thus improving the plant’s ability to withstand environmental challenges.
Bra026262 is potentially involved in the sexual reproduction of B. rapa, as indicated by its high expression levels in flowers and decreased expression in female sterile mutants. At4g32480 (homologs: Bra026262 and Bra024209) may interact with MYB59, a member of the MYB family, and AGL56, a MADS box protein. MYB family members such as MYB64/119 [33], FLP and MYB88 [34], and MYB98 [35] are known to regulate synergid cell differentiation, pollen tube guidance, and the formation of the filiform apparatus during female gametophyte development. MADS box proteins, including AGL51, AGL52, and AGL78, play roles in the female gametophyte [36]. Additionally, AGL9 and AGL15 recruit the FIS-PRC2 complex to regulate endosperm proliferation by modulating H3K27me3 marks and gene expression, suggesting a conserved role for MADS-box proteins in PRC2-mediated development across plants [37]. These findings suggest that Bra026262 may interact with MYB and AGL proteins, potentially recruiting the FIS-PRC2 complex to regulate female gametophyte growth and development.

4. Materials and Methods

4.1. Identification of DUF506 Members in B. rapa

The genome data file for B. rapa was retrieved from the BRAD database. (http://brassicadb.cn/), and the A. thaliana whole genome sequences were obtained from TAIR (https://www.Arabidopsis.org/), and the candidate BrDUF506 members were identified through a two-way BLAST search of the B. rapa genome. Thirteen Arabidopsis DUF506 protein sequences were downloaded from the UniProtKB database (https://www.uniprot.org), and the eighteen BrDUF506 protein sequences were obtained from EnsemblPlants (http://plants.ensembl.org/), used as queries to perform protein blast searches by using TBtools [38]. Meantime, the typical domain of DUF506 (PDDEXK_6) was obtained from the Pfam database (http://pfam.xfam.org/) and was used to search for DUF506 with the HMMER tool (https://www.ebi.ac.uk/). The molecular weight (MW) and isoelectric point (pI) of BrDUF506 members were predicted with ExPASy (http://web.expasy.org/protparam/). Subcellular localization data were predicted with WoLF PSORT (Protein Subcellular Localization Prediction, https://wolfpsort.hgc.jp/).

4.2. Chromosomal Distribution, Phylogenetic Relationship, and Collinearity Analysis

The distribution of BrDUF506 genes on the B. rapa chromosome was determined from B. rapa gff3 annotation files obtained from BRAD and plotted using TBtools software (v2.119). While the chromosome density was calculated using the Gene Density Profile plug-in in TBtools (v2.119) with default settings. The naming of BrDUF506s was based on their respective chromosomal locations.
Several Plug-ins, such as Advanced Circos, the Dual Synteny, and Table Row extract or filter in TBtools (v2.119) software, were used for synteny analysis. Collinearity relationships between duplicate genes within B. rapa and between A. thaliana and O. sativa were analyzed and visualized by TBtools.
Phylogenetic trees for the DUF506 genes in B. rapa and A. thaliana were generated through maximum likelihood estimation utilizing the OmicShare platform. (https://www.omicshare.com/) by setting bootstrap to 1000, and further modified by using iTOL (https://itol.embl.de/) tools.

4.3. Conserved Motif, Gene Structure, and Cis-Element Prediction of BrDUF506 Family Genes

The conserved motifs were analyzed and predicted with MEME (http://meme-suite.org/) and visualized by TBtools (v2.119). Visualization of the gene structure of BrDUF506s was achieved through the application of the TBtools basic Visual Gene Structure program.
The BrDUF506s’ promoters were obtained through EnsemblPlants Family gene initiation codon 2000 bp upstream sequences and used to search for cis-elements with the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) with default parameters.

4.4. Gene Expression Analysis

BrDUF506s tissue-specific expression data in different tissues (callus, flower, leaf, root, silique, and stem tissues) were extracted from BRAD (http://brassicadb.cn/) and visualized using TBtools (v2.119). AtDUF506s expression data under various abiotic stress treatments were downloaded from the Arabidopsis eFP Browser (http://bar.utoronto.ca/). For significant data, heatmap matrices were plotted using TBtools.
The transcriptome data of B. rapa in two important mutants associated with sexual reproduction (male sterility mutant, msm, and female sterility mutant, fsm) was obtained from NCBI GEO (https://www.ncbi.nlm.nih.gov/geo/) with accession numbers GSE125485 and GSE147438 and normalized using the transcripts per million (TPM) method. Gene expression heatmaps were plotted using TBtools.

4.5. Plant Growth Conditions, Abiotic Stress Treatments, and Transcriptome

B. rapa-cultivated species with stable self-incompatibility was used for expression analysis. The plump seeds were sown in MS medium and cultivated in a plant incubator. Six leafy seedlings were selected for abiotic stress treatments. The seedlings were grown in a hydroponic system with 150 mM NaCl to simulate salt stress and in 15% PEG6000 for drought conditions. The seedlings were exposed to 4 °C for cold stress and 28 °C to simulate heat stress. At the same time, used similar growth status untreated B. rapa seedlings as a control (CK). All materials were collected for RNA extraction and transcriptome analysis. A minimum of three biological replicates were performed for each treatment.

4.6. Prediction of Protein-Protein Interaction Networks

Predictions for the protein-protein interaction network analysis were performed utilizing the STRING website (https://cn.string-db.org/) with default parameters, and Cytoscape v3.10.2 was used to construct the interaction network.

5. Conclusions

In conclusion, the comprehensive analysis of genome-wide identification, expression profiling, and protein-protein interactions suggests that Bra000098 and Bra017099 are potential regulators of heat and drought stress tolerance, while Bra026262 is involved in the regulation of female gametophyte growth and development (Figure 9). These findings provide valuable insights into the functional roles of these genes in B. rapa, which is essential for devising strategies to increase crop resilience and inform breeding practices. Through the application of gene knockout or overexpression techniques, we can precisely gauge the impact of these genes on stress resistance and reproductive functions, underscoring their potential in crop improvement. These genes could be promising targets for marker-assisted selection in breeding programs aimed at enhancing crop resilience. The targeted identification and selection of gene variants that correlate with enhanced stress tolerance can enable breeders to cultivate crop varieties with improved capabilities to endure rigorous environmental stressors. In summary, the results presented in this study have the potential to significantly advance our understanding of gene function in Brassica rapa and to drive innovation in crop breeding, ultimately contributing to the development of more resilient and high-yielding varieties.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms252011087/s1.

Author Contributions

X.Z., X.Y. and J.H. conceived the project and designed the experiments; G.Z. and J.W. performed the bioinformatics analysis and experiments with help from S.H., K.L. and R.Z. G.Z. and J.W. wrote the manuscript. X.Z., X.Y. and J.H. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Hunan Provincial Natural Science Foundation (2024JJ6129).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All the data that support the findings of this study are available in this paper and its Supplementary Material published online.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Bateman, A.; Coggill, P.; Finn, R.D. DUFs: Families in search of function. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2010, 66 Pt 10, 1148–1152. [Google Scholar] [CrossRef] [PubMed]
  2. Urbanowicz, B.R.; Pena, M.J.; Ratnaparkhe, S.; Avci, U.; Backe, J.; Steet, H.F.; Foston, M.; Li, H.; O’Neill, M.A.; Ragauskas, A.J.; et al. 4-O-methylation of glucuronic acid in Arabidopsis glucuronoxylan is catalyzed by a domain of unknown function family 579 protein. Proc. Natl. Acad. Sci. USA 2012, 109, 14253–14258. [Google Scholar] [CrossRef] [PubMed]
  3. Mewalal, R.; Mizrachi, E.; Coetzee, B.; Mansfield, S.D.; Myburg, A.A. The Arabidopsis Domain of Unknown Function 1218 (DUF1218) Containing Proteins, MODIFYING WALL LIGNIN-1 and 2 (At1g31720/MWL-1 and At4g19370/MWL-2) Function Redundantly to Alter Secondary Cell Wall Lignin Content. PLoS ONE 2016, 11, e0150254. [Google Scholar] [CrossRef]
  4. Li, Y.; Huang, Y.; Wen, Y.; Wang, D.; Liu, H.; Li, Y.; Zhao, J.; An, L.; Yu, F.; Liu, X. The domain of unknown function 4005 (DUF4005) in an Arabidopsis IQD protein functions in microtubule binding. J. Biol. Chem. 2021, 297, 100849. [Google Scholar] [CrossRef]
  5. Chen, K.; Wang, Y.; Nong, X.; Zhang, Y.; Tang, T.; Chen, Y.; Shen, Q.; Yan, C.; Lu, B. Characterization and in silico analysis of the domain unknown function DUF568-containing gene family in rice (Oryza sativa L.). BMC Genom. 2023, 24, 544. [Google Scholar] [CrossRef]
  6. Kaur, V.; Yadav, S.K.; Wankhede, D.P.; Pulivendula, P.; Kumar, A.; Chinnusamy, V. Cloning and characterization of a gene encoding MIZ1, a domain of unknown function protein and its role in salt and drought stress in rice. Protoplasma 2020, 257, 475–487. [Google Scholar] [CrossRef]
  7. Xin, Z.; Mandaokar, A.; Chen, J.; Last, R.L.; Browse, J. Arabidopsis ESK1 encodes a novel regulator of freezing tolerance. Plant J. 2007, 49, 786–799. [Google Scholar] [CrossRef] [PubMed]
  8. Sakata, M.; Takano-Kai, N.; Miyazaki, Y.; Kanamori, H.; Wu, J.; Matsumoto, T.; Doi, K.; Yasui, H.; Yoshimura, A.; Yamagata, Y. Domain Unknown Function DUF1668-Containing Genes in Multiple Lineages Are Responsible for F(1) Pollen Sterility in Rice. Front. Plant Sci. 2020, 11, 632420. [Google Scholar] [CrossRef] [PubMed]
  9. Wang, W.; Xiong, H.; Zhao, P.; Peng, X.; Sun, M.X. DMP8 and 9 regulate HAP2/GCS1 trafficking for the timely acquisition of sperm fusion competence. Proc. Natl. Acad. Sci. USA 2022, 119, e2207608119. [Google Scholar] [CrossRef]
  10. Knizewski, L.; Kinch, L.N.; Grishin, N.V.; Rychlewski, L.; Ginalski, K. Realm of PD-(D/E)XK nuclease superfamily revisited: Detection of novel families with modified transitive meta profile searches. BMC Struct. Biol. 2007, 7, 40. [Google Scholar] [CrossRef]
  11. Ying, S.; Scheible, W.R. A novel calmodulin-interacting Domain of Unknown Function 506 protein represses root hair elongation in Arabidopsis. Plant Cell Environ. 2022, 45, 1796–1812. [Google Scholar] [CrossRef] [PubMed]
  12. Ying, S. Genome-Wide Identification and Transcriptional Analysis of Arabidopsis DUF506 Gene Family. Int. J. Mol. Sci. 2021, 22, 11442. [Google Scholar] [CrossRef]
  13. Dong, W.; Tu, J.; Deng, W.; Zhang, J.; Xu, Y.; Gu, A.; An, H.; Fan, K.; Wang, R.; Zhang, J.; et al. Genome-wide identification of DUF506 gene family in Oryzasativa and expression profiling under abiotic stresses. PeerJ 2023, 11, e16168. [Google Scholar] [CrossRef] [PubMed]
  14. Fedoroff, N.V.; Battisti, D.S.; Beachy, R.N.; Cooper, P.J.; Fischhoff, D.A.; Hodges, C.N.; Knauf, V.C.; Lobell, D.; Mazur, B.J.; Molden, D.; et al. Radically rethinking agriculture for the 21st century. Science 2010, 327, 833–834. [Google Scholar] [CrossRef] [PubMed]
  15. Qiao, X.; Li, Q.; Yin, H.; Qi, K.; Li, L.; Wang, R.; Zhang, S.; Paterson, A.H. Gene duplication and evolution in recurring polyploidization-diploidization cycles in plants. Genome Biol. 2019, 20, 38. [Google Scholar] [CrossRef] [PubMed]
  16. Danino, Y.M.; Even, D.; Ideses, D.; Juven-Gershon, T. The core promoter: At the heart of gene expression. Biochim. Biophys. Acta 2015, 1849, 1116–1131. [Google Scholar] [CrossRef]
  17. Sych, T.; Levental, K.R.; Sezgin, E. Lipid-Protein Interactions in Plasma Membrane Organization and Function. Annu. Rev. Biophys. 2022, 51, 135–156. [Google Scholar] [CrossRef]
  18. Arora, B.; Tandon, R.; Attri, P.; Bhatia, R. Chemical Crosslinking: Role in Protein and Peptide Science. Curr. Protein Pept. Sci. 2017, 18, 946–955. [Google Scholar] [CrossRef]
  19. Hsu, K.H.; Liu, C.C.; Wu, S.J.; Kuo, Y.Y.; Lu, C.A.; Wu, C.R.; Lian, P.J.; Hong, C.Y.; Ke, Y.T.; Huang, J.H.; et al. Expression of a gene encoding a rice RING zinc-finger protein, OsRZFP34, enhances stomata opening. Plant Mol. Biol. 2014, 86, 125–137. [Google Scholar] [CrossRef]
  20. Huang, Y.; Liu, L.; Hu, H.; Tang, N.; Shi, L.; Xu, F.; Wang, S. Arabidopsis ERF012 Is a Versatile Regulator of Plant Growth, Development and Abiotic Stress Responses. Int. J. Mol. Sci. 2022, 23, 6841. [Google Scholar] [CrossRef]
  21. Zizelski Valenci, G.; Raveh, D.; Bar-Zvi, D. The activity of the stress modulated Arabidopsis ubiquitin ligases PUB46 and PUB48 is partially redundant. Plant Signal Behav. 2022, 17, 2072111. [Google Scholar] [CrossRef] [PubMed]
  22. Peng, L.; Wan, X.; Huang, K.; Pei, L.; Xiong, J.; Li, X.; Wang, J. AtPUB48 E3 ligase plays a crucial role in the thermotolerance of Arabidopsis. Biochem. Biophys. Res. Commun. 2019, 509, 281–286. [Google Scholar] [CrossRef] [PubMed]
  23. Brasil, J.N.; Cabral, L.M.; Eloy, N.B.; Primo, L.M.; Barroso-Neto, I.L.; Grangeiro, L.P.; Gonzalez, N.; Inze, D.; Ferreira, P.C.; Hemerly, A.S. AIP1 is a novel Agenet/Tudor domain protein from Arabidopsis that interacts with regulators of DNA replication, transcription and chromatin remodeling. BMC Plant Biol. 2015, 15, 270. [Google Scholar] [CrossRef] [PubMed]
  24. He, S.; Zhi, F.; Min, Y.; Ma, R.; Ge, A.; Wang, S.; Wang, J.; Liu, Z.; Guo, Y.; Chen, M. The MYB59 transcription factor negatively regulates salicylic acid- and jasmonic acid-mediated leaf senescence. Plant Physiol. 2023, 192, 488–503. [Google Scholar] [CrossRef] [PubMed]
  25. Fasani, E.; DalCorso, G.; Costa, A.; Zenoni, S.; Furini, A. The Arabidopsis thaliana transcription factor MYB59 regulates calcium signalling during plant growth and stress response. Plant Mol. Biol. 2019, 99, 517–534. [Google Scholar] [CrossRef]
  26. Rasheed, S.; Bashir, K.; Kim, J.M.; Ando, M.; Tanaka, M.; Seki, M. The modulation of acetic acid pathway genes in Arabidopsis improves survival under drought stress. Sci. Rep. 2018, 8, 7831. [Google Scholar] [CrossRef]
  27. Gan, Y.; Filleur, S.; Rahman, A.; Gotensparre, S.; Forde, B.G. Nutritional regulation of ANR1 and other root-expressed MADS-box genes in Arabidopsis thaliana. Planta 2005, 222, 730–742. [Google Scholar] [CrossRef]
  28. Du, B.; Haensch, R.; Alfarraj, S.; Rennenberg, H. Strategies of plants to overcome abiotic and biotic stresses. Biol. Rev. Camb. Philos. Soc. 2024, 99, 1524–1536. [Google Scholar] [CrossRef]
  29. Martienssen, R.A. Heterochromatin, small RNA and post-fertilization dysgenesis in allopolyploid and interploid hybrids of Arabidopsis. New Phytol. 2010, 186, 46–53. [Google Scholar] [CrossRef]
  30. Yang, J.L.; Chen, W.W.; Chen, L.Q.; Qin, C.; Jin, C.W.; Shi, Y.Z.; Zheng, S.J. The 14-3-3 protein GENERAL REGULATORY FACTOR11 (GRF11) acts downstream of nitric oxide to regulate iron acquisition in Arabidopsis thaliana. New Phytol. 2013, 197, 815–824. [Google Scholar] [CrossRef]
  31. Urzica, E.I.; Casero, D.; Yamasaki, H.; Hsieh, S.I.; Adler, L.N.; Karpowicz, S.J.; Blaby-Haas, C.E.; Clarke, S.G.; Loo, J.A.; Pellegrini, M.; et al. Systems and trans-system level analysis identifies conserved iron deficiency responses in the plant lineage. Plant Cell 2012, 24, 3921–3948. [Google Scholar] [CrossRef] [PubMed]
  32. Liu, X.X.; Zhu, X.F.; Xue, D.W.; Zheng, S.J.; Jin, C.W. Beyond iron-storage pool: Functions of plant apoplastic iron during stress. Trends Plant Sci. 2023, 28, 941–954. [Google Scholar] [CrossRef] [PubMed]
  33. Rabiger, D.S.; Drews, G.N. MYB64 and MYB119 are required for cellularization and differentiation during female gametogenesis in Arabidopsis thaliana. PLoS Genet. 2013, 9, e1003783. [Google Scholar] [CrossRef] [PubMed]
  34. Makkena, S.; Lee, E.; Sack, F.D.; Lamb, R.S. The R2R3 MYB transcription factors FOUR LIPS and MYB88 regulate female reproductive development. J. Exp. Bot. 2012, 63, 5545–5558. [Google Scholar] [CrossRef]
  35. Kasahara, R.D.; Portereiko, M.F.; Sandaklie-Nikolova, L.; Rabiger, D.S.; Drews, G.N. MYB98 is required for pollen tube guidance and synergid cell differentiation in Arabidopsis. Plant Cell 2005, 17, 2981–2992. [Google Scholar] [CrossRef]
  36. Cai, H.; Liu, L.; Zhang, M.; Chai, M.; Huang, Y.; Chen, F.; Yan, M.; Su, Z.; Henderson, I.; Palanivelu, R.; et al. Spatiotemporal control of miR398 biogenesis, via chromatin remodeling and kinase signaling, ensures proper ovule development. Plant Cell 2021, 33, 1530–1553. [Google Scholar] [CrossRef]
  37. Zhang, S.; Mohanty, D.; Muzaffar, A.; Ni, M. Two MADS-box proteins, AGL9 and AGL15, recruit the FIS-PRC2 complex to trigger the phase transition from endosperm proliferation to embryo development in Arabidopsis. Mol. Plant 2024, 17, 1110–1128. [Google Scholar] [CrossRef]
  38. Chen, C.; Wu, Y.; Li, J.; Wang, X.; Zeng, Z.; Xu, J.; Liu, Y.; Feng, J.; Chen, H.; He, Y.; et al. TBtools-II: A “one for all, all for one” bioinformatics platform for biological big-data mining. Mol. Plant 2023, 16, 1733–1742. [Google Scholar] [CrossRef]
Ijms 25 11087 g001
Figure 1. Chromosome localization and phylogenetic analysis of BrDUF506 genes. (A) Chromosome localization of BrDUF506s, with chromosomes indicated in green bars, and black lines out of the colored box show BrDUF506s distribution in chromosomes. (B) Phylogenetic tree of DUF506 gene family in B. rapa and A. thaliana. Green, purple, pink, and yellow correspond to Groups I, II, IIIa, and IIIb, respectively.
Figure 1. Chromosome localization and phylogenetic analysis of BrDUF506 genes. (A) Chromosome localization of BrDUF506s, with chromosomes indicated in green bars, and black lines out of the colored box show BrDUF506s distribution in chromosomes. (B) Phylogenetic tree of DUF506 gene family in B. rapa and A. thaliana. Green, purple, pink, and yellow correspond to Groups I, II, IIIa, and IIIb, respectively.
Ijms 25 11087 g001
Ijms 25 11087 g002
Figure 2. Synteny analysis of DUF506s in B. rapa, A. thaliana, and O. sativa. (A) Gray bars indicate the chromosomes of B. rapa. The orange lines in boxes represent the gene density of the chromosomes. Colored lines suggest duplicated gene pairs of B. rapa. (B) Chromosomal collinearity relationships between B. rapa, A. thaliana, and O. sativa. Orange, blue, and green indicate B. rapa, A. thaliana, and O. sativa, respectively. Red lines suggest collinear gene pairs.
Figure 2. Synteny analysis of DUF506s in B. rapa, A. thaliana, and O. sativa. (A) Gray bars indicate the chromosomes of B. rapa. The orange lines in boxes represent the gene density of the chromosomes. Colored lines suggest duplicated gene pairs of B. rapa. (B) Chromosomal collinearity relationships between B. rapa, A. thaliana, and O. sativa. Orange, blue, and green indicate B. rapa, A. thaliana, and O. sativa, respectively. Red lines suggest collinear gene pairs.
Ijms 25 11087 g002
Ijms 25 11087 g003
Figure 3. Gene structure and conserved motifs of BrDUF506s. (A) The phylogenetic tree of DUF506 family in B. rapa; four different background colors indicate four subfamilies. (B) Exon-intron structure. Green rectangles and black lines indicate exons and introns, respectively. (C) Composition and distribution of conserved motifs in BrDUF506s. Motifs are shown by 18 different color bars.
Figure 3. Gene structure and conserved motifs of BrDUF506s. (A) The phylogenetic tree of DUF506 family in B. rapa; four different background colors indicate four subfamilies. (B) Exon-intron structure. Green rectangles and black lines indicate exons and introns, respectively. (C) Composition and distribution of conserved motifs in BrDUF506s. Motifs are shown by 18 different color bars.
Ijms 25 11087 g003
Ijms 25 11087 g004
Figure 4. Distribution of cis-acting elements in BrDUF506s promoters.
Figure 4. Distribution of cis-acting elements in BrDUF506s promoters.
Ijms 25 11087 g004
Ijms 25 11087 g005
Figure 5. BrDUF506s are involved in abiotic stress tolerance. (A) Analysis of BrDUF506 gene transcriptome data under heat treatment. Seedlings of B. rapa with six leaves were exposed to 28 °C lasting for 6 h before collection. (B) Analysis of BrDUF506 gene transcriptome data under drought treatment. Seedlings of B. rapa with six leaves were subjected to a 15% PEG6000 solution to mimic drought conditions, with treatment lasting for 6 h before collection. Unstressed B. rapa seedlings with the same growth period and under identical growth conditions were used as a control (CK). All seedlings were harvested for RNA extraction and subsequent transcriptome analysis. Each group contained three biological replicates. Data are means ± SD; significant differences were determined by Student’s t test, ** p < 0.01.
Figure 5. BrDUF506s are involved in abiotic stress tolerance. (A) Analysis of BrDUF506 gene transcriptome data under heat treatment. Seedlings of B. rapa with six leaves were exposed to 28 °C lasting for 6 h before collection. (B) Analysis of BrDUF506 gene transcriptome data under drought treatment. Seedlings of B. rapa with six leaves were subjected to a 15% PEG6000 solution to mimic drought conditions, with treatment lasting for 6 h before collection. Unstressed B. rapa seedlings with the same growth period and under identical growth conditions were used as a control (CK). All seedlings were harvested for RNA extraction and subsequent transcriptome analysis. Each group contained three biological replicates. Data are means ± SD; significant differences were determined by Student’s t test, ** p < 0.01.
Ijms 25 11087 g005
Ijms 25 11087 g006
Figure 6. Tissue expression heatmap of BrDUF506s. All values underwent logarithmic transformation. Deeper pink, larger dots indicate higher expression level; deeper blue, larger dots indicate lower expression level.
Figure 6. Tissue expression heatmap of BrDUF506s. All values underwent logarithmic transformation. Deeper pink, larger dots indicate higher expression level; deeper blue, larger dots indicate lower expression level.
Ijms 25 11087 g006
Ijms 25 11087 g007
Figure 7. Expressions of BrDUF506 in different sexual reproduction-related mutants. Analysis of the transcriptome data of BrDUF506s expression values in male sterile mutants (msm) (A) and female sterile mutants (fsm) (B) compared with wild-type (FT). The heatmap demonstrates the expression level; the color gradient from white to purple presents increasing expression values.
Figure 7. Expressions of BrDUF506 in different sexual reproduction-related mutants. Analysis of the transcriptome data of BrDUF506s expression values in male sterile mutants (msm) (A) and female sterile mutants (fsm) (B) compared with wild-type (FT). The heatmap demonstrates the expression level; the color gradient from white to purple presents increasing expression values.
Ijms 25 11087 g007
Ijms 25 11087 g008
Figure 8. The PPI networks of DUF506 proteins in B. rapa. (A) The predicted Abiotic stress-related Bra017099 and Bra000098 PPIs. (B) The predicted sexual reproduction-related Bra026262 PPIs.
Figure 8. The PPI networks of DUF506 proteins in B. rapa. (A) The predicted Abiotic stress-related Bra017099 and Bra000098 PPIs. (B) The predicted sexual reproduction-related Bra026262 PPIs.
Ijms 25 11087 g008
Ijms 25 11087 g009
Figure 9. The putative molecular mechanism underlying the regulation of stress tolerance and sexual reproduction by BrDUF506s.
Figure 9. The putative molecular mechanism underlying the regulation of stress tolerance and sexual reproduction by BrDUF506s.
Ijms 25 11087 g009
Table 1. Characteristics of the BrDUF506 gene family.
Table 1. Characteristics of the BrDUF506 gene family.
Gene IDChromosome (Chr)StartEndpIMolecular Weight
(Average)		Subcellular LocationProtein Length (aa)A. thaliana
DUF506 Genes
Bra026262A0110,328,54410,330,2396.9633,655.44nucl299AT4G32480
Bra036877A0112,460,52612,461,7425.6939,571.58cyto354AT4G14620
Bra040373A0127,947,91327,948,49110.7821,600.19chlo192AT3G07350
Bra000098A039,236,5609,237,6568.3834,659.74nucl309AT2G38820
Bra000140A039,501,6749,503,5527.0433,728.35nucl302AT2G39650
Bra001897A0319,213,11019,214,1895.5536,930.09cyto333AT3G22970
Bra013223A0319,776,41319,777,2616.5531,935.97nucl282AT3G25240
Bra024209A0326,866,82426,868,1366.9933,163.73nucl295AT4G32480
Bra017099A0416,615,63016,616,6038.733,762.84nucl300AT2G38820
Bra033896A0514,926,74014,927,9795.3440,567.86cyto367AT3G22970
Bra029641A0522,850,13322,851,0356.5433,558.79nucl300AT3G07350
Bra019736A064,719,3934,720,9206.2133,768.01cyto293AT1G12030
Bra036492A0788,23489,5976.933,380.91nucl297AT2G20670
Bra015691A0721,336,47421,337,5458.7630,140.59nucl263AT1G77145
Bra015690A0721,338,68121,339,7548.6930,837.18nucl268AT1G77145
Bra015689A0721,345,30621,346,3188.7629,586.01nucl257AT1G77145
Bra016797A0820,033,88020,035,8217.5633,604.24cyto295AT1G12030
Bra031136A0932,332,83532,334,1247.5333,184.72nucl, cyto_nucl294AT2G20670
aa, amino acids. Subcellular location: chlo (chloroplast), cyto (cytosol), nucl (nucleus).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Zhu, G.; Wang, J.; He, S.; Liang, K.; Zhang, R.; Huang, J.; Yang, X.; Zhang, X. Comprehensive Analysis of BrDUF506 Genes Across the Brassica rapa Genome Uncovers Potential Functions in Sexual Reproduction and Abiotic Stress Tolerance. Int. J. Mol. Sci. 2024, 25, 11087. https://doi.org/10.3390/ijms252011087

AMA Style

Zhu G, Wang J, He S, Liang K, Zhang R, Huang J, Yang X, Zhang X. Comprehensive Analysis of BrDUF506 Genes Across the Brassica rapa Genome Uncovers Potential Functions in Sexual Reproduction and Abiotic Stress Tolerance. International Journal of Molecular Sciences. 2024; 25(20):11087. https://doi.org/10.3390/ijms252011087

Chicago/Turabian Style

Zhu, Guangqi, Jingxuan Wang, Shuang He, Kexin Liang, Renyi Zhang, Jiabao Huang, Xueqin Yang, and Xiaojing Zhang. 2024. "Comprehensive Analysis of BrDUF506 Genes Across the Brassica rapa Genome Uncovers Potential Functions in Sexual Reproduction and Abiotic Stress Tolerance" International Journal of Molecular Sciences 25, no. 20: 11087. https://doi.org/10.3390/ijms252011087

APA Style

Zhu, G., Wang, J., He, S., Liang, K., Zhang, R., Huang, J., Yang, X., & Zhang, X. (2024). Comprehensive Analysis of BrDUF506 Genes Across the Brassica rapa Genome Uncovers Potential Functions in Sexual Reproduction and Abiotic Stress Tolerance. International Journal of Molecular Sciences, 25(20), 11087. https://doi.org/10.3390/ijms252011087

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop