Genome-wide and expression analysis to understand the DUF789 gene family during development of Arabidopsis thaliana

1 Introduction

Advancements in sequencing technology have led to an exponential increase in data related to genomics, transcriptomics, proteomics, and metabolomics. Despite the vast amount of data that has been generated, a significant portion of it remains unexplored (Chaudhari et al., 2024). There exists a group of conserved protein families that are composed of domains with unknown functions that have not yet been characterized. In 1998, researcher Chris Ponting was the first person to identify and designate these domains as DUF1 and DUF2 (Vishwakarma et al., 2024). After sequencing genomes in various species, additional DUF families were subsequently identified. Additionally, the Pfam database version 35.0 includes a total of 19,632 families, with 4,795 of them classified as DUF families. Certain proteins that possess DUFs play a crucial role in plant development. These proteins include DUF724, DUF1218, and DUF231 (Lv et al., 2023). Cellulose comprises the primary and secondary cell walls. Proteins containing the DUF266 domain are believed to play a role in cellulose biosynthesis, possibly functioning as glycosyltransferases. A mutation in the DUF266 gene significantly decreases cellulose synthesis in rice (Yang et al., 2017). The A. thaliana possesses six genes related to RUS, all of which code for proteins featuring the DUF647 domain. However, both RUS1 and RUS2 have been reported to have a significant role in regulating early seedling growth, vitamin B6 homeostasis and auxin transport (Tong et al., 2021). Furthermore, the expression of RUS6 was observed during various stages of plant growth, with a notably high level of expression in flowers. This indicates the significant role of RUS6 in the development of A.thaliana (Perry et al., 2021). Research conducted by Vergès et al. (2023) discovered that 23 genes were found to be expressed in the reproductive organs, of A. thaliana with the majority of them being expressed in the endosperm (Vergès et al., 2023). In A. thaliana DUF239 genes show a characteristic expression pattern that suggests a new role for plant neprosin-related proteins, particularly during seed maturation. Furthermore several studies have reported on the role of DUF gene families in A. thaliana and rice in responding to abiotic stress (Zhong et al., 2019). In A. thaliana, the TBL3 and TBR genes encode proteins that contain the DUF231 domain. This domain has significant function in the development of secondary cell walls in plants (Yuan et al., 2016). In A. thaliana the ESK1 gene (DUF231), acts as an inhibitor during cold acclimation (Yuan et al., 2013). Furthermore, the expression of the AtRDUF1 and AtRDUF2 genes in A. thaliana was found to be stimulated by abscisic acid (ABA) and drought stress. Conversely, when their expression is decreased, it leads to reduced drought stress tolerance. The salt-responsive gene TaSRHP (DUF581) was overexpressed in transgenic A. thaliana plants, resulting in increased resistance to stress (Hou et al., 2013). Furthermore, overexpression of the TaSRG (DUF662) transcription factor has been shown to enhance salt tolerance in both rice and A. thaliana. However, the gene SbSGL (DUF1645) has a significant function in sorghum by regulating the process of seed maturation (Zhang Bin et al., 2018). A mutation in the DUF1517 gene of A. thaliana renders it susceptible to cold stress (HAO et al., 2018). In this study, we analyzed the AtDUF789s at the genome-wide level. Our analysis included conducting a phylogenetic analysis, examining the gene structure, and assessing the chromosome location. Furthermore,we have identified members of the DUF789 family in several plant species including G. max, Vitis vinifera, Solanum tuberosum, Medicago truncatula, and Sorghum bicolor in this study. Moreover, we conducted an analysis of the expression of AtDUF789s in various plant tissues, including leaves, flowers, fruits, and roots, utilizing RNA-seq data. This study provides a basis for future research on the role of AtDUF789 genes. Further investigation into AtDUF789 genes will also enhance our understanding of the regulatory mechanism of DUF789s in crops.

2 Materials and Methods

3 Results

3.2

3.2 Cis-Regulatory element analysis

Specifically, this research examined eight cis-regulatory elements, four of which are related to phytohormones: gibberellin, methyl jasmonate, auxin, and abscisic acid. Furthermore, the components related to phytohormones include TGA, ABRE, TATC box, GARE motif, P-box, CGTCA motif, and TGACG motif. These components were found in various genes, emphasizing the crucial role of AtDUF789s in regulating phytohormones. Furthermore, we discovered components that respond to different types of stresses, namely anaerobic, low-temperature, drought, and salt. These components consist of the TCA-component, MBS, LTR, and ARE, indicating their involvement in stress response. Specifically, the MBS component, which is responsible for responding to drought, was predominantly discovered in AtDUF789-6, AtDUF789-7, and AtDUF789-8 (Supplementary Table S2).

The low-temperature responsive (LTR) element was found in six genes, namely AtDUF789-2, AtDUF789-3, AtDUF789-4, AtDUF789-5, AtDUF789-8, and AtDUF789-11. The anaerobic responsiveness (ARE) component was present in AtDUF789-1, AtDUF789-2, AtDUF789-3, AtDUF789-4, AtDUF789-5, AtDUF789-6, AtDUF789-7, AtDUF789-8, AtDUF789-9, AtDUF789-10, and AtDUF789-11 (Fig. 1).

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Fig. 1

Cis-elements in the promoter regions of the AtDUF789 genes are linked with different hormone- and stress-responsive elements. Different color boxes show different identified elements.

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3.3

3.3 Construction of phylogenetic tree for DUF789 genes

A phylogenetic tree for DUF789 was created using protein sequences from A. thaliana, G. max, Vitis vinifera, Solanum tuberosum, Medicago truncatula, and Sorghum bicolor. The tree was divided into 5 groups, as shown in Fig. 2. Group I contained 15 proteins (4 S. bicolor, 4 M. truncatula, 2 S. tuberosum, 3 V. vinifera, and 2 G. max) (Fig. 2). Group II consisted of 15 proteins (2 M. truncatula, 1 S. tuberosum, 4 V. vinifera, 6 G. max, and 2 A. thaliana). Group III included 16 proteins (4 S. bicolor, 2 M. truncatula, 2 S. tuberosum, 2 V. vinifera, 4 G. max, and 2 A. thaliana). Group IV comprised 20 proteins (3 S. bicolor, 3 M. truncatula, 4 S. tuberosum, 4 A. thaliana, 4 G. max, and 2 Vitis vinifera). Group V consisted of 13 proteins (3 A. thaliana, 4 G. max, 1 V. vinifera, 3 S. tuberosum, and 2 M. truncatula). According to the constructed tree, Group IV had the highest number of proteins, while Group V had the lowest.

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Fig. 2

A neighbor-joining phylogenetic tree assessment of DUF789 genes from S. tuberosum, G. max, Sorghum bicolor, A. thaliana,Vitis vanifera and M. truncatula. Overall, AtDUF789 were clustered into five major classes.

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3.4

3.4 Analysis of conserved motifs and domains

Ten unique motifs were identified and their locations were analyzed using the MEME tool for AtDUF789 proteins. Additionally, the full amino acid sequences were analyzed to identify conserved motifs. It was observed that all AtDUF789 proteins contain an AtDUF789 superfamily domain, indicating that this domain is conserved across all proteins.

During the motif analysis of AtDUF789 genes, it was found that genes within the same classes may contain different motifs, which could act as regulators for the various activities of the classes. For example, motif 1, with 50 amino acids (Supplementary Table S1), was found to be unique to group I, while motif 10 was also found in AtDUF789-3 and AtDUF789-4. The majority of genes have motifs 1, 4, and 6. It was also observed that motif 8 was unique to certain genes. For instance, motif 8 alone was present in GmDUF789-1, GmDUF789-6, GmDUF789-8, GmDUF789-7, and GmDUF789-11, while other motifs such as 1, 2, 3, 4, 5, 6, 7, 8, and 9 were identified in GmDUF789-1, GmDUF789-6, and GmDUF789-7 (Fig. 3).

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Fig. 3

The gene structure and motif analysis of AtDUF789 genes. Based on phylogenetic relationships, the AtDUF789 were clustered into five major classes. (A) Conserved motif compositions were detected in AtDUF789. Different color boxes represent different motifs. (B) Gene structure of AtDUF789 genes. The light yellow color denotes exon and the black horizontal line symbolizes introns.

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3.6

3.6 Chromosomal positions and synteny analysis of AtDUF789s

The chromosomal positions of all 11 AtDUF789 genes indicate that the genes were unequally distributed across chromosomes. Table 1 provides the specific chromosome positions for the AtDUF789 genes. The results show that four chromosomes contain the AtDUF789 genes, with chromosomes 1, 2, 4, and 5 all having AtDUF789 genes. In total, there were eleven AtDUF789 genes across these four chromosomes, with three genes located in chromosomes 4 and 5 (Fig. 4).

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Fig. 4

Chromosomal distribution of AtDUF789 genes. The green color showed Chromosomes. Red color genes.

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Additionally, this investigation reveals the presence of synteny among Vitis vinifera, S. tuberosum, G. max, A. thaliana, S. bicolor, and M. truncatula plants. The purpose of this investigation was to determine the role, evolution, expression, and duplications of DUF789 genes. Our results indicate a synteny relationship between the sequences VIT_205s0077g01510, AtDUF789-11, and GmDUF789-7. Furthermore, AtDUF789-2 and AtDUF789-5 of A. thaliana and the gene VIT_201s0011g04000 of M. truncatula also exhibited synteny. Notably, AtDUF789-6 of A. thaliana showed synteny with the S. tuberosum gene PGSC0003DMG400021665 sequence (Fig. 5).

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Fig. 5

Synteny map among all identified DUF789 sequences of S. tuberosum, M. truncatula, A. thaliana, and G. max.

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3.8

3.8 Expression of AtDUF789s in different tissues

RNA-seq data were used to examine the expression of AtDUF789s in leaf, flower, fruit, and root tissues. The analysis revealed that several AtDUF789 genes, namely AtDUF789-1, AtDUF789-2, AtDUF789-3, AtDUF789-4, AtDUF789-5, AtDUF789-6, AtDUF789-7, AtDUF789-8, AtDUF789-9, AtDUF789-10, and AtDUF789-11, showed high expression levels in leaf, flower, fruit, and root tissues (Fig. 6). For instance, AtDUF789-1 had high expression in both leaves and roots, while AtDUF789-6 showed high expression in flower and root tissues (Supplementary Table S5). These genes were found to be highly expressed throughout plant development, along with other genes.

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Fig. 6

Expression profiling of AtDUF789 genes in various developmental tissues. The red,white, and green colors display high to low expression. levels.

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4 Discussion

The DUF gene family is known to regulate various biological functions in plants (Zaynab et al., 2022). However, there has not been a thorough investigation of the AtDUF789 gene family in A. thaliana. In this study, we aim to fill this gap by conducting a genome-wide identification of DUF789 genes. Additionally, we examined the functional evolution of different members of the DUF789 gene family in the Arabidopsis thaliana genome. Furthermore, the current study has discovered a total of 11 AtDUF789s within the genome of Arabidopsis thaliana. In order to identify the distinctive qualities of the AtDUF789s within the family, we also analyzed the physicochemical features of the eleven members. This analysis revealed that each AtDUF789 possesses its own unique characteristics. Zaynab et al. (2023) reported that each GmDUF668 protein exhibited unique characteristics (Zaynab et al., 2023b). We conducted subcellular localization prediction to determine the specific location of AtDUF789 proteins within intracellular organelles. Upon examination of subcellular localization, it was found that the majority of AtDUF789 proteins were primarily located within the nucleus (Paulo et al., 2013). This suggests that these proteins play a crucial role in this organelle.

Exons play a crucial role in the protein synthesis process by containing essential information. On the other hand, introns have a protective function, preventing specific random mutations that could potentially harm the coding proteins (Lesk, 2010). The analysis has significant implications for researchers who study the evolutionary perspective, structure, and function of the DUF789 gene family. Furthermore, the examination of the gene structure has unveiled the quantity of exons and introns in the AtDUF789 genes. This analysis of gene structure yields crucial insights into the evolutionary patterns of exon–intron distribution and the factors that influence the diverse range of biochemical functions (Gelfman et al., 2012). Furthermore, it was observed that various AtDUF789s shared the same structure. Furthermore the analysis of the gene structure of AtDUF789 revealed variations in the number of exons and introns. Moreover, the differences in the number of exons observed among the AtDUF789 genes imply functional variations within this gene family. Similarly, the analysis of conserved gene motifs suggests that some AtDUF789 genes share the same set of motifs. The fact that the AtDUF789 genes have similar motifs suggests that they are evolutionarily related and serve the same biological purpose.

The promoters of AtDUF789s contain cis-acting components that demonstrate their responsiveness to different hormones and stresses. This suggests that AtDUF789s can be regulated by various stresses (salinity, drought, and low temperature) as well as phytohormones (auxin, ABA, MeJA, and GA). The current study findings are supported by multiple studies that have reported the role of cis-components in a plant's response to stress (Raza et al., 2020). In addition, TaGAPC1 also responds to various stresses and its expression pattern is controlled in response to salinity and osmotic stress. During stress, the LTR, GT1, DRE, and MBS elements of the TaGAPC1 promoter undergo alterations due to methylation (Wen et al., 2021). This modification serves to counterbalance the effects of stress and regulate the gene's expression levels. MicroRNAs, also known as miRNAs, are a group of small RNAs that are non-coding and endogenous in nature. They typically measure 20–22 nucleotides in size (Ling et al., 2013). These miRNAs play a significant role in various cellular and physiological processes, including plant development, growth, and response to environmental stress conditions (Kumar, 2014). Several studies have shown that miRNAs play a potential role in various biological processes. In this study, we identified several miRNAs that target 11 AtDUF789 genes. Importantly, our findings are in line with previous research and serve to confirm our results. Wang et al. (2020) examined the role of miR156 in A. thaliana (Ye et al., 2020), while Wang et al. (2020) focused on investigating the developmental role of miR156 in rice (Wang et al., 2023). Additionally, Zhao et al. 2024 reported that miR172s play a positive regulatory role in development (Zhao et al., 2024). Previous studies have also confirmed the significance of miR172 and its target expression in plant developmental processes. Spanudakis et al and Jackson reported the essential role of miR159 at different stages of plant growth (Spanudakis and Jackson, 2014). Therefore, these findings suggest that miRNAs play a vital role in plant development, growth, and stress tolerance.

To investigate the involvement of AtDUF789 genes in plant development, we examined the expression of 11 specific genes in leaves, flowers, fruits, and roots. This analysis was carried out using an RNA-seq dataset from the BioProject PRJNA168212. Furthermore, previous studies have demonstrated that the expression patterns of certain DUF genes undergo significant variations across different tissue types throughout the process of development. A study conducted by Zhang et al. (2024) revealed that OsDUF247 genes were expressed in rice (Zhang et al., 2024). Their findings revealed that OsDUF247 was expressed in seedlings, roots, stems, and leaves. Zaynab et al. (2023) conducted a study on the expression of DUF668 genes in soybean (Zaynab et al., 2023b). Their findings revealed that GmDUF668 was expressed in flowers, nodules, roots, and leaves. The expression analysis demonstrated higher levels of gene expression in these tissues. Specifically, genes such as GmDUF668-30, GmDUF668-28, GmDUF668-27, GmDUF668-26, GmDUF668-22, GmDUF668-19, GmDUF668-17, GmDUF668-16, GmDUF668-15, GmDUF668-13, GmDUF668-10, GmDUF668-9, GmDUF668-8, GmDUF668-7, GmDUF668-4, and GmDUF668-3, showed upregulation across roots, nodules, leaves, and flowers (Zaynab et al., 2023b). The expression of GhDUF4228 genes in various tissues of cotton, including stem, root, leaf, bract, pistil, sepal, petal, anther, and filament was also investigated. The gene expression profiles at different developmental stages provided information about various genes (Rochette et al., 2008). It was reported that certain genes exhibited upregulation in different parts of the plant, including the stem, root, leaf, bract, pistil, sepal, petal, anther, and filament (Rochette et al., 2008). Furthermore, Aulakh et al. (2014) conducted a tissue-specific expression analysis for sweet potato using RNA-seq data (Aulakh et al., 2014). The findings revealed that several IbDUF668 genes had increased expression patterns across different tissues during development. Multiple IbDUF668 genes were found to exhibit increased expression in tissues during development. Upon examining the transcriptome data for the GmDUF4228 genes, it was found that these genes were expressed in leaves, root hairs, nodules, and pods. It is worth noting that GmDUF4228-56, GmDUF4228-70, and GmDUF4228-73 exhibit particularly strong expression in roots (Leng et al., 2021). The study found that several DUF599 genes were expressed in potato tissues. To assess the importance of StDUF599-6 and StDUF599-9 in development, their expression in tissues was examined (Zaynab et al., 2023a). These findings support that AtDUF789s play a crucial role in plant development.

5 Conclusions

In this investigation, we identified a total of 11 AtDUF789s. These AtDUF789s were classified into five clusters based on a phylogenetic tree. To create this tree, we used protein sequences from G.max, Vitis vinifera, Solanum tuberosum, Medicago truncatula, Sorghum bicolor, and A.thaliana. Furthermore, the proteins ranged from 186 to 409, and with molecular weights between 21.15 and 45.81 kDa. Analysis of cis-elements suggests that AtDUF789 genes may be involved in stress responses. Most of the identified AtDUF789 genes have shown significant upregulation during the expression stages. This suggests that AtDUF789s play a crucial role in plant development. Further analysis of DUF789 genes will yield valuable data for the molecular breeding of A. thaliana, aiming to improve plant growth.

CRediT authorship contribution statement

Madiha Zaynab: Conceptualization, Data curation, Formal analysis. Yasir Sharif: Investigation, Methodology. Rashid Al-Yahyai: Software, Supervision, Validation. Athar Hussain: Writing – original draft. Monther Sadder: Writing – review & editing. Kahkashan Perveen: Data curation, Formal analysis. Najat A. Bukhari: Methodology. Shuangfei Li: Writing – review & editing.