Skip to main content
Download PDF

Genome-wide identification and transcriptome profiling expression analysis of the U-box E3 ubiquitin ligase gene family related to abiotic stress in maize (Zea mays L.)

BMC Genomics volume 25, Article number: 132 (2024) Cite this article

Abstract

Background

The U-box gene family encodes E3 ubiquitin ligases involved in plant hormone signaling pathways and abiotic stress responses. However, there has yet to be a comprehensive analysis of the U-box gene family in maize (Zea mays L.) and its responses to abiotic stress.

Results

In this study, 85 U-box family proteins were identified in maize and were classified into four subfamilies based on phylogenetic analysis. In addition to the conserved U-box domain, we identified additional functional domains, including Pkinase, ARM, KAP and Tyr domains, by analyzing the conserved motifs and gene structures. Chromosomal localization and collinearity analysis revealed that gene duplications may have contributed to the expansion and evolution of the U-box gene family. GO annotation and KEGG pathway enrichment analysis identified a total of 105 GO terms and 21 KEGG pathways that were notably enriched, including ubiquitin-protein transferase activity, ubiquitin conjugating enzyme activity and ubiquitin-mediated proteolysis pathway. Tissue expression analysis showed that some ZmPUB genes were specifically expressed in certain tissues and that this could be due to their functions. In addition, RNA-seq data for maize seedlings under salt stress revealed 16 stress-inducible plant U-box genes, of which 10 genes were upregulated and 6 genes were downregulated. The qRT-PCR results for genes responding to abiotic stress were consistent with the transcriptome analysis. Among them, ZmPUB13, ZmPUB18, ZmPUB19 and ZmPUB68 were upregulated under all three abiotic stress conditions. Subcellular localization analysis showed that ZmPUB19 and ZmPUB59 were located in the nucleus.

Conclusions

Overall, our study provides a comprehensive analysis of the U-box gene family in maize and its responses to abiotic stress, suggesting that U-box genes play an important role in the stress response and providing insights into the regulatory mechanisms underlying the response to abiotic stress in maize.

Peer Review reports

Background

Plant growth is frequently challenged by various abiotic stresses, including salt, drought, cold and heat, exerting a substantial negative impact on global crop productivity. It is estimated that up to 50% of global crop losses can be attributed to abiotic stress, with more than 90% of these losses due to salt, drought and temperature stress [1]. Amidst the intricate molecular responses to abiotic stress, protein ubiquitination has become a focal point of investigation due to its dynamic alterations in response to stress stimuli [2].

The ubiquitin–proteasome pathway is one of the major protein degradation pathways in nuclear organisms [3]. In the ubiquitination pathway, the binding of ubiquitin to the target protein is mediated by a three-step enzyme cascade. The protein is catalyzed by the ubiquitin-activating enzyme E1, ubiquitin-binding enzyme E2s and ubiquitin ligase E3s [4, 5], and the protein can be recognized and degraded by the proteasome after labeling with polyubiquitin. E3 ubiquitin ligases are the largest family in ubiquitin proteasome degradation mechanisms, which include diverse types such as the RING, F-box, HECT and U-box [6, 7]. Studies have shown that RING and U-box type E3s are widely involved in abiotic stress processes, while the F-box protein is the core component of SKP1-Cullin1-F-box protein type E3 and plays an important role in the abiotic stress response [8]. The U-box protein structure, which functions as a ubiquitin ligase, is a conserved motif comprising approximately 70 amino acids. It is prevalent in fungi, plants, and animals, and it frequently interacts with ARM repeats in plants. This interaction influences protein–protein interactions and plays a crucial role in signal transduction cascades [9].

Crucially, E3 ubiquitin ligases, particularly U-box proteins, have been identified as key regulators enhancing abiotic stress tolerance in major food crops. For instance, in rice, OsPUB15 acts as a negative regulator of cell death and abiotic stress responses, and its overexpression enhances salt tolerance in plants [10]. Moreover, OsPUB41 plays a crucial role as a negative regulator in modulating drought stress tolerance [11]. Similarly, U-box proteins in wheat, including TaPUB1 and TaPUB15, exert positive effects on salt stress tolerance [12, 13], while TaPUB26 acts as a negative regulator in response to salt stress [14] In maize, ZmAIRP4 enhances drought stress tolerance [15], and ZmRFP1 responds to drought stress in an ABA-dependent manner [16]. These examples underscore the intricate involvement of U-box ubiquitin ligases in the modulation of abiotic stress responses across diverse plant species. Furthermore, U-box ubiquitin ligases associated with abiotic stress have been identified in various plant species, including soybean, tomato, potato, pepper, apple and Arabidopsis, where they play crucial roles in regulating drought tolerance, salt tolerance and ABA signaling [17,18,19,20,21,22].

The U-box gene family has been identified in numerous plants, and the number of genes varies among species. In crop plants, U-box genes have been identified in the genomes of various plants, including rice, cabbage, soybean, tomato, barley, cotton (G. barbadense), wheat, sorghum, potato, Brassica oleracea L., wild emmer wheat and Solanum tuberosum, with a total of 41 to 213 genes, respectively [23,24,25,26,27,28,29,30,31,32,33,34]. In fruit plants such as banana, stony hard peach, white pear, apple and strawberry, with a total of 62 to 765 U-box genes have been identified, respectively [35,36,37,38,39].

Despite the important role of E3 ubiquitin ligase in abiotic stress responses in model plants such as Arabidopsis, rice and wheat, the understanding of their involvement in maize remains limited. Maize, one of the most widely grown crops in the world, often faces challenges such as salinity, drought, cold and heat stress. Drought alone inflicts an annual economic impact of $20 billion on maize yields [15]. Consequently, enhancing the stress resistance of maize plants is crucial for achieving higher yields. Despite numerous individual maize genes have been analyzed, a comprehensive study of the maize genome in response to abiotic stress is currently lacking. Therefore, it is essential to explore the role of U-box genes in stress signaling and to clarify their contribution to maize growth and development. These studies will provide a theoretical foundation for comprehending stress response mechanisms and provide a basis for selecting and breeding improved maize varieties with enhanced tolerance. The insights derived from this study promise a deeper understanding of the functions of the maize U-box gene family, paving the way for advancements in crop resilience and agricultural productivity.

Results

Identification of ZmPUB family genes in maize

To identify U-box family genes, we used the U-box domain (PF04564) to perform bioinformatic searches in the maize genome database using HMMER 3.0. The accuracy of the candidate U-box genes was checked using the Pfam database with an E-value cutoff level of 1.0. A total of 85 ZmPUB genes were confirmed and renamed ZmPUB1-85 based on the order of their gene IDs (Table 1). The molecular weights of the 85 ZmPUB proteins ranged from 10 to 150 kDa, and the predicted amino acid quantities ranged from 90 to 1400. Among the 85 ZmPUB proteins, 57 ZmPUB proteins were acidic proteins with theoretical isoelectric points ranging from 4.65 to 6.97. There were 21 ZmPUB proteins with theoretical isoelectric points between 8.12 and 9.36 and are basic proteins. The other 7 ZmPUB proteins were electroneutral, with theoretical isoelectric points ranging from 7.01 to 7.93. The amino acid length and protein characteristics of these proteins were evidently different, suggesting that ZmPUB family members have different characteristics and play different roles in biological processes. Subcellular localization prediction results showed that most of the genes were located in the nucleus, and a few genes were predicted to be located in both the cytoplasm and the nucleus.

Table 1 Basic information of U-box family genes in maize
Full size table

Phylogenetic relationship of PUB proteins in maize, rice and Arabidopsis

To investigate the evolutionary relationships among PUB genes, we constructed a neighbor-joining (NJ) phylogenetic tree using U-box protein sequences from maize, rice and Arabidopsis (85 from maize, 77 from rice and 61 from Arabidopsis) (Table S1, Table S2 and Table S3). The topology of the phylogenetic tree was divided into four subfamilies. Notably, Group 4 exhibited the highest number of PUB genes, encompassing a total of 73, while Group 2 contained the fewest PUB genes, numbering only 11. Additionally, Group 1 and Group 3 consisted of 57 and 59 genes, respectively (Fig. 1).

Fig. 1
figure 1

Evolutionary relationship analysis of the U-box protein family in maize, Arabidopsis thaliana and Oryza sativa. Phylogenetic tree of the U-box gene family in maize, Arabidopsis and rice. Multiple sequence alignments for U-box domain sequences of 85 ZmPUBs, 61 AtPUBs and 77 OsPUBs were conducted via MEGA 7.0. The phylogenetic tree was established using the neighbor-joining method with MEGA7.0 software, employing the p-distance method and a 1000-bootstrap value. The resulting PUB genes were categorized into four distinct groups (G1-G4), each identified by a unique color

Full size image

Although the domains were not unequivocally categorized within the phylogenetic groups, it was evident that each group contained ZmPUBs, all of which featured the U-box domain. Notably, Group 4, the largest group, comprised 31 ZmPUBs, 25 OsPUBs and 17 AtPUBs and was distinguished by the presence of 20 ARM domains. The ZmPUB proteins within the phylogenetic tree were predominantly associated with Prp19 domain-containing proteins, primarily within the G3 subfamily. On the other hand, Pkinase, Pkinase Tyr, Ufd2P core and Usp domain-containing proteins were primarily found within the G1 subfamily (Fig. 1). These associations underscore the relationship between the phylogeny and evolution of ZmPUB proteins and suggest that the U-box protein domain has coevolved alongside other functional regions. In general, the PUB genes of maize and rice exhibited a closer evolutionary relationship to each other than to those of Arabidopsis.

Functional domain analysis of the U-box proteins

The SMART and Pfam database inquiries revealed that the U-box proteins contained several known or unknown conserved domains, which are essential for functionality and have been designated as functional domains (Fig. 2). Some U-box proteins had no other obvious interaction domains or had a few rare functionally uncertain domains, collectively categorized as 'Unknown' (only have U-box). Among the 85 U-box protein families, 37 genes had no other obvious functional domains except U-box (Unknown), 21 genes contain the Arm domain, 8 genes contain the Pkinase domain, and 4 genes contain the Prp19 domain. In addition, other functional domains were detected, such as WD40, Tyr, Ufd2P core, Usp, ANAPC3, DWNN, KAP, Myb DNA-bind3, Neurochondrin and zf-CCHC2. The ARM repeat is a series repeat motif approximately 40 amino acids long that binds to S-site receptor kinase (SRK) and is an important part of the downstream SRK signaling pathway [40]. Studies have shown that the U-box protein PHOR1 of potato (Solanum tuberosum) with these ARM repeat may play a role in gibberellin signaling [41]. These domains are essential for plant defense and disease resistance.

Fig. 2
figure 2

Number and domain structure of U-box proteins in maize. The domain names are taken from the Pfam database, and the position of the domain markers also refers to the position shown in the database. The different types of domains are marked with graphs of different colors and shapes, and a representative gene is provided for each domain. "Unknown" designates that, apart from the U-box domain, no other evident functional domains were identified

Full size image

Chromosome localization and collinearity analysis of ZmPUBs

According to the information on the physical positions of ZmPUBs in maizeGDB, the 85 ZmPUB genes, except for two genes that did not appear on the nucleus, were distributed randomly on the 10 chromosomes in maize, with the most genes on chromosome 1 and the fewest genes on chromosome 8 (Fig. 3A and Table S4). The results showed that ZmPUBs are mainly concentrated at both ends of the chromosome. The uneven distribution of the 83 ZmPUB genes shows the complex diversity of U-box family genes in maize. In addition, chromosomes 2, 5 and 9 have concentrated gene clusters.

Fig. 3
figure 3

Chromosomal localizations and gene duplications of the ZmPUB family genes (A), collinearity analysis with Arabidopsis and rice (B-D). A The scale represents megabases (Mb), and the chromosome number is shown at the top of each bar. The protein sequences encoded by straight-line junction genes are more than 70% similar. B Collinearity relationships of U-box genes in maize. C Collinear relationships between U-box genes in maize and Arabidopsis thaliana. D Collinearity relationships between U-box genes in maize and rice. Blue lines represent collinear gene pairs of the PUB gene family. The inner circular heatmap represents gene density. The outer circles with scales represent chromosomes

Full size image

The gene duplication events were analyzed to reveal the expansion mechanism of the ZmPUB gene family. We found 23 gene pairs involved in gene duplication events. Previous studies defined gene duplication event based on two criteria: alignment sequence length covering ≥ 80% of the longest genes and sequence similarity ≥ 70% within the alignment region [42]. Among these duplicated genes, there were three gene pairs with more than 95% similarity: ZmPUB16/ZmPUB64, ZmPUB12/ZmPUB30 and ZmPUB80/ZmPUB51. Furthermore, since ZmPUB53/ZmPUB38 are on the same chromosome and the physical locations of ZmPUB81/ZmPUB18 and ZmPUB59/ZmPUB43 are similar, it can be concluded that ZmPUB53/ZmPUB38 are not crossed on the chromosome.

The gene collinearity results showed a total of 61 tandem duplicate gene pairs on the 10 chromosomes in the maize genome (Fig. 3B and Table S5). Sixty-two collinear PUB gene pairs were screened in maize and rice (Fig. 3C and Table S6), while only 3 collinear PUB gene pairs were found in Arabidopsis and maize (Fig. 3D and Table S7). In Arabidopsis, chromosomes 2, 3 and 4 do not show the collinear U-box gene. In rice, the collinearity of the U-box gene was not shown on chromosomes 7 and 11, but there were different amounts of U-box gene collinearity on the other chromosomes.

The rates of non-synonymous (Ka) and synonymous (Ks) substitutions serve as the basis for evaluating the selective pressure in repetitive events. A Ka/Ks ratio of 1 indicates neutral selection, Ka/Ks < 1 suggests purifying selection, and Ka/Ks > 1 implies positive selection. The Ka/Ks ratios for tandem duplications in ZmPUB genes were computed using KaKs Calculator 3.0 [43], revealing a range of 0.0547 to 0.6840, with an average of 0.2760 (Fig. S1, Table S8). All Ka/Ks values for duplicated events were less than 1, suggesting that these genes predominantly evolved under purifying selection. Additionally, we calculated the potential dates of duplication events, which occurred between 13.79 and 313.42 million years ago (Mya).

For further exploration of the potential evolutionary processes in the ZmPUB gene family, we analyzed the synteny relationships of ZmPUB genes in rice, Arabidopsis and maize. The Ka/Ks range for Arabidopsis and maize was 0.0606 to 0.6422, with an average of 0.2023. In rice and maize, the Ka/Ks range was 0.0281 to 0.8655, with an average of 0.1776.

Conservative motif and structural analysis of the U-box gene family in maize

All the identified U-box genes were analyzed online based on MEME for the presence of novel and undetermined motifs, and 20 conserved motifs of maize U-box proteins were identified (Fig. 4A). U-box proteins exhibit a variable number of motifs, with certain members, like ZmPUB84, possessing up to 14 motifs. Some proteins contain a few motifs, among which the ZmPUB1 protein contains only 1 motif. Further analysis revealed that 80 ZmPUB proteins contained Motif 1 and 45 ZmPUB proteins contained Motif 6. Motif 1 was found in proteins containing Motif 6, suggesting that Motif 1 is related to Motif 6. The majority of U-box proteins contain Motif 1, 2 and 3, with Motif 1 accounting for 94% (80), Motif 2 for 89% (76), and Motif 3 for 83.5% (71). These findings suggest that these three motifs play an important role in the U-box gene family. It is also noticeable that Motif 1, 3 and 2 are always adjacent and appear at both ends of the sequence. Motif 5 is always adjacent and appears after Motif 19. Most of the adjacent ZmPUB proteins ( In the same clade within the evolutionary tree and the position is adjacent), including ZmPUB64 and ZmPUB16, ZmPUB59 and ZmPUB43, and ZmPUB83 and ZmPUB21, contain the same motifs. These gene pairs have the same motif type, number and position.

Fig. 4
figure 4

Analysis of the conserved motifs and gene structure of the U-box family in maize. A The phylogenetics, distribution of conserved motifs. The Neighbor-Joining (NJ) phylogenetic tree of 85 maize PUB genes, which were constructed using the MEGA7 software (Neighbor-Joining, NJ) with the bootstrap parameter set to 1000. The subfamily information is represented by G1-G4. Based on MEME online analysis, the 20 conserved motifs of the U-box proteins in maize were designated motifs 1 ~ 20 and are represented by different colors. B Distribution of gene structure. The exon/intron structure of ZmPUB genes was determined by comparing CDS with genomic sequences. Green and yellow rectangles represents untranslated region (UTR) and coding DNA sequence (CDS) features, respectively, the horizontal line represents gene regions, and gray lines represents introns

Full size image

The ZmPUB gene structure was analyzed by comparing the CDS and genomic DNA sequences of the ZmPUB gene (Fig. 4B). The results showed notable differences in gene structure between the ZmPUB genes, with the number of introns ranging from 1 to 17 (Table S9). Within the U-box protein family, thirty-one of the 85 genes in total have no introns, suggesting that the evolution of these genes may follow the transposon mechanism. As shown in Fig. 4B, ZmPUB83, ZmPUB21, ZmPUB49 and ZmPUB18 did not contain introns. ZmPUB64 and ZmPUB16 contain up to 17 introns. The exon length and arrangement order of the ZmPUB47 and ZmPUB77 genes were similar, but the intron length was different.

Cis-regulatory element analysis in the promoter regions of U-box genes

The cis-regulatory elements upstream of the start codon are essential for transcriptional regulation of protein-coding genes. U-box genes play an important role in the response to various stresses. Cis-acting elements located in the promoter region of genes can regulate the expression of related genes by binding transcription factors. To further investigate the relationship between ZmPUB genes and stress, we used the PlantCARE database to predict the potential cis-acting elements of the putative ZmPUBs promoter region (Fig. 5).

Fig. 5
figure 5

Analysis of the cis-regulatory elements in the promoter regions of ZmPUB genes. Different colors represent different cis-regulatory elements located within 1500 bp upstream of ZmPUBs, including ARE, ATC motif, DRE1, GATT motif, G-box, GC motif, I-box, LS7, ARBE, O2-site, OTC and TATA-box

Full size image

An analysis of the 1500 bp upstream region of the ZmPUB gene revealed the presence of 12 cis-acting elements. These elements encompass crucial regulatory components, including stress-related elements (ARE, DRE1, ATC-motif, GATT-motif, G-box, GC-motif, I-box, LS7), hormone-related elements (ABRE, O2-site), and defense and pressure-related elements (OCT, TATA-box). It is noteworthy that all ZmPUB genes contain cis-acting elements associated with hormone regulation, with a majority linked to stress or hormonal regulation.

We categorized these cis-regulatory elements into three distinct groups based on their involvement in related biological processes: the growth and development process, encompassing two types of elements; phytohormone response, comprising two types of elements; and stress response and includes eight types of elements (Fig. 5 and Table S10). Remarkably, out of the total 373 elements, a substantial majority (71%, 266 elements) belonged to the stress-responsive group. Among these stress-responsive elements, ARE was the most prevalent member, accounting for 39%, while the GATT motif was less frequent, with only one occurrence. In the phytohormone response group, the majority (72%) of the elements are ABREs, implying the likely involvement of ZmPUB genes in phytohormone responses. In contrast, the growth and development group exhibited the fewest elements, with only seven in total. Additionally, on average, each U-box gene contained four cis-acting elements. ZmPUB12 and ZmPUB61 contained the most cis-acting elements, with 10. It seems that the presence of these elements suggested that ZmPUB genes could be transcriptionally regulated by abiotic stresses.

Gene ontology annotation and KEGG pathway enrichment of the maize U-box gene

To gain a deeper understanding of the biological function of the U-box gene, we conducted GO annotation and KEGG pathway enrichment analysis (Fig. 6). The results show that 105 GO terms covering biological processes, cellular components and molecular functions were notably enriched. Notably, molecular functions were diverse and included key activities such as ubiquitin-protein transferase activity, ubiquitin-conjugating enzyme activity, signal receptor binding and transmembrane receptor protein-serine/threonine kinase binding (Fig. 6A and Table S11). These functions play a crucial role in protein interactions, signal transduction and the regulation of various biological processes in cells. The biological processes involved mainly include the response to organonitrogen compounds, the response to chitin, protein autoubiquitination and protein polyubiquitination. The most prevalent cellular components include the Prp19 complex, the U2 spliceosome complex and the Cul4-RING E3 ubiquitin ligase complex.

Fig. 6
figure 6

Gene Ontology enrichment and KEGG pathway analysis of the U-box gene in maize. A GO enrichment shown as dot bubbles. A total of 105 GO terms were notably enriched, covering biological processes, cellular components, and molecular functions. B KEGG enrichment shown as a bar graph. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to verify the specific metabolic pathways related to these genes

Full size image

We performed a comprehensive analysis of the U-box gene to investigate its functionality in metabolic and signal transduction pathways using eggnog-mapper. The results show that the U-box gene contributes notably to 21 pathways, including ubiquitin-mediated proteolysis, Cushing syndrome, spliceosome and protein processing in the endoplasmic reticulum (Fig. 6B and Table S12). In particular, the ubiquitin-mediated proteolysis pathway was the most represented, with nine U-box genes.

Expression analysis of the U-box protein-encoding genes in different tissues

To explore the function of U-box genes in different tissues, we were able to examine the expression of ZmPUB genes in 22 tissues using an existing database (https://www.maizegdb.org/expression). These tissues included the ear, internode, kernel, leaf, reproductive tissue and root (Fig. 7 and Table S13). Some ZmPUB genes were expressed only in certain tissues. For example, ZmPUB45, ZmPUB47, ZmPUB72 and ZmPUB73 were expressed in mature pollen; ZmPUB32, ZmPUB35, ZmPUB57 and ZmPUB42 were mainly expressed in roots; ZmPUB41, ZmPUB43 and ZmPUB53 were mainly expressed in kernels; and ZmPUB21 and ZmPUB59 were mainly expressed in kernels and reproductive tissue. In the ear group, ZmPUB6, ZmPUB10, ZmPUB13, ZmPUB18, ZmPUB25, ZmPUB31, ZmPUB33, ZmPUB49, ZmPUB64, ZmPUB71 and ZmPUB84 had high expression levels, whereas ZmPUB13 and ZmPUB49 were expressed only in the longer ear primordium. Some ZmPUB genes, such as ZmPUB15, ZmPUB19, ZmPUB27, ZmPUB44, ZmPUB48, ZmPUB53, ZmPUB65, ZmPUB66, ZmPUB67 and ZmPUB74, were barely expressed in the 22 tissues and may have more specific functions. Because the ZmPUB gene plays a crucial role in the response to stress, plant growth and development, expression pattern analysis can provide important clues for the study of function.

Fig. 7
figure 7

Expression of the U-box protein family genes in each tissue. Different colors at the top represent different groups (Ear, Internode, Kernel, Leaf, Root and Reproductive), and each of the following columns represents 22 tissues. The standardized FPKM values are shown on the top right scale, with red representing high expression and blue representing low expression

Full size image

Expression pattern of ZmPUB genes under abiotic stress

We conducted gene expression analysis of U-box family genes in maize under salt stress at 2, 12 and 24 h with 0-h treatment as a control (Fig. S2). Genes were considered to have differences in expression levels if |log2N|≧1. The correlation analysis of all transcriptome samples showed a difference between the control and treatment groups, and the differences deepened significantly with treatment time (Fig. 8A). After salt treatment, ZmPUB13, ZmPUB18, ZmPUB19, ZmPUB41, ZmPUB59, ZmPUB60, ZmPUB63, ZmPUB68, ZmPUB79 and ZmPUB83 were upregulated (considered upregulated if their expression increased at 2, 12 and 24 h with a log2 fold change > 1 for all three time points). ZmPUB3, ZmPUB42, ZmPUB47, ZmPUB50, ZmPUB56 and ZmPUB58 were downregulated (considered downregulated if their expression decreased at 2, 12 and 24 h with a log2 fold change < -1 for all three time points) (Fig. 8B and Table S14).

Fig. 8
figure 8

Correlation analysis of the salt stress samples across time periods and expression heatmap of the ZmPUB genes. A Correlation analysis between samples exposed to 0, 2, 12 and 24 h of salt stress treatments. Color intensity reflects the strength of the correlation, with darker colors representing stronger correlations. B Expression heatmap of ZmPUB genes under 0, 2, 12 and 24 h of salt stress treatment. The x-axis denotes different samples of salt stress, while the y-axis represents ZmPUB genes ordered based on hierarchical cluster analysis. The color scale in the upper right corner corresponds to the standardized log2 fold changes (log2FC) values. Genes were considered upregulated if their expression increased at 2, 12, and 24 h with a log2 fold change > 1 for all three time points, and downregulated if their expression decreased at these time points with a log2 fold change < -1. “ + ” representing up-regulated and “-” representing down-regulated

Full size image

To verify the high-throughput sequencing data, 8 genes with relatively high up-regulation or down-regulation responding to salt stress were selected for qRT-PCR experiments and were consistent with the transcriptome analysis (Fig. 9). Under salt stress, ZmPUB13, ZmPUB18, ZmPUB19, ZmPUB59, ZmPUB60 and ZmPUB68 were upregulated, while ZmPUB38 and ZmPUB45 were notably downregulated. Among them, the transcription levels of ZmPUB19 and ZmPUB59 increased notably, and the upregulation rates reached tenfold and 80-fold, respectively, after 12 h of salt stress treatment. After 24 h of salt stress treatment, ZmPUB59 was upregulated 120-fold. Considering that the U-box genes were induced by more than one stress condition, we conducted qRT-PCR to examine the 8 genes under drought and heat stress conditions. Under drought stress, ZmPUB13, ZmPUB18, ZmPUB19, ZmPUB59, ZmPUB60 and ZmPUB68 were notably upregulated, while ZmPUB38 and ZmPUB45 were notably downregulated in the early stage ( 2h and 12h) of treatment (Fig. 9). Under heat stress, ZmPUB13, ZmPUB18, ZmPUB19, ZmPUB38 and ZmPUB68 were notably upregulated, while ZmPUB59 and ZmPUB60 were notably downregulated after 12 h of treatment. The results showed that ZmPUB13, ZmPUB18, ZmPUB19 and ZmPUB68 were induced by salt, drought and heat treatments. Furthermore, ZmPUB38 and ZmPUB45 were downregulated during the early stages of salt and drought treatments, but they were upregulated under heat stress.

Fig. 9
figure 9

qRT-PCR analysis of the U-box gene under salt, drought and high temperature stress. The 4-week-old seedlings were subjected to salt, drought and high temperature stress for 0, 2, 12 and 24 h. Eight abiotic stress-responsive genes were selected for qRT-PCR experiments, and the results were consistent with the RNA-seq data: ZmPUB13, ZmPUB18, ZmPUB19, ZmPUB59, ZmPUB60 and ZmPUB68 were upregulated, while ZmPUB38 and ZmPUB45 were notably downregulated. Some U-box genes were induced by more than one stress condition. For example, ZmPUB13, ZmPUB18, ZmPUB19 and ZmPUB68 were induced by salt, drought and heat treatments

Full size image

Subcellular localization of ZmPUB19 and ZmPUB59

To further elucidate the biological function of the ZmPUB gene in maize under abiotic stress, we selected the two genes, ZmPUB19 and ZmPUB59, which exhibited the highest upregulation under salt stress conditions according to the results of qRT-PCR and RNA-seq, for subcellular localization analysis. The 35S:ZmPUB19-GFP or 35S:ZmPUB59-GFP fusion expression vector and the nuclear marker protein (NLS-mcherry) were coexpressed in tobacco leaves (Nicotiana benthamiana) by Agrobacterium-mediated osmosis. The results showed strong fluorescence expression signals of 35S:ZmPUB19-GFP and 35S:ZmPUB59-GFP and were consistent with the expression signals of the nuclear marker protein (NLS-mcherry) (Fig. 10), thus confirming that ZmPUB19 and ZmPUB59 are mainly localized in the nucleus.

Fig. 10
figure 10

Analysis of the subcellular localization of ZmPUB19 and ZmPUB59. ZmPUB19-GFP and ZmPUB59-GFP colocalized with NLS-mCherry. Paired constructs were infected into Nicotiana leaves. GFP and mCherry fluorescence signals were monitored 40–48 h after infection by confocal microscopy. For each construct, 10–20 cells were analyzed, and a similar nuclear localization pattern was observed. ZmPUB19-GFP and ZmPUB59-GFP had strong fluorescence expression signals in the nucleus and coincided with the NLS-mcherry expression signal in the nucleus, confirming that ZmPUB19 and ZmPUB59 were mainly localized in the nucleus. Scale bar, 20 μm

Full size image

Discussion

Abiotic stresses such as salinity, drought and high temperature, profoundly impact maize growth [44]. The ubiquitin pathway emerges as a critical player in posttranslational protein modifications, with U-box proteins, functioning as E3 ligases, play essential roles in plant growth and stress responses. Despite that several studies have focused on individual maize U-box genes and RNA-seq has been conducted to assess the impact of hormones (ABA, IAA, and GA) on the expression of ZmPUB [45], a comprehensive genome-level analysis of the maize U-box protein family's response to abiotic stress has been lacking.

In this study, we identified a total of 85 ZmPUB genes in maize (Table 1), classified into four subfamilies through phylogenetic analysis (Fig. 1). Additional functional domains were discovered in 85 U-box protein families (Fig. 2). These domains are essential for plant defense and disease resistance. Previous studies have shown that the ARM domain is the second most conserved domain in rice and Arabidopsis and is associated with substrate recognition in the ubiquitination process. In maize, 21 ZmPUB proteins have the ARM domain and is similar to those in rice and Arabidopsis. ARM repeats are characteristic repetitive amino acid sequences identified in proteins, with a length of approximately 40 residues, and proteins containing these sequences typically have multiple tandem copies [46, 47]. In general, E3 ubiquitin ligases have protein–protein interaction domains that interact with their substrates to facilitate ubiquitination [48]. For instance, the ARM repeat protein AtPUB-ARM in Arabidopsis regulates various cellular processes [49]. PUB-ARM genes have been reported to respond to diverse abiotic stresses in different plant species. Cho et al. found that the PUB-ARM gene was affected by salinity stress in hot pepper [50]. In Arabidopsis, two PUB-ARM genes, AtPUB18 and 19, were found to be transiently expressed under ABA treatment [51]. In cabbage, the homologous BrPUB-ARM genes BrPUB6 and BrPUB54 were upregulated under ABA treatment. These findings emphasize the important role of PUB genes in response to various environmental pressures.

Gene duplication (GD), mutation and natural selection are the main sources of new genes with novel functions and provide the foundation for biological diversity [52]. Chromosomal analysis revealed random distribution of the 85 ZmPUB genes across maize chromosomes, with tandem duplicates and collinearity observed. The association between maize PUB genes and rice PUB genes is stronger compared to Arabidopsis and is consistent with the results of homologous evolution analysis between maize and rice (Fig. 3). It is possible that homologous PUB genes perform similar functions in maize and rice. Tandem repeats, as a major component of U-box gene extensions, can effectively contribute to maintaining gene structure and function. This may also be a mechanism for acquiring new functional domains in genes [53]. The whole-genome duplication (WGD) has significant implications for species evolution, as it generates new gene copies, contributing significantly to adaptability, isolation, phenotypic robustness, and evolution. These events result in the emergence of numerous complex gene families in descendant species [54]. In addition to multigene duplications, there are also numerous single-gene duplications. Although these single-gene duplications often result in gene loss [55], the retained genes have important implications for evolution [56]. The analysis of the Ka/Ks ratios for tandem duplications in ZmPUB genes revealed values predominantly less than 1, indicating that these genes primarily evolved under purifying selection. Notably, the comparison of the Ka/Ks ratios between Arabidopsis and maize, and rice and maize, showed lower values for the latter pairs. This suggests that maize U-box genes may have experienced stronger purifying selection as compared to their counterparts in Arabidopsis and rice. The lower Ka/Ks values for rice and maize may reflect a more stringent elimination of deleterious mutations, potentially contributing to the conservation and functional stability of the U-box gene family in these species. This suggests that the U-box gene family in maize has undergone relatively stronger selective pressure, possibly contributing to the high functional conservation of these genes. Consequently, these findings point to the potential impact of selective pressures on the evolutionary processes and functional characteristics of the U-box gene family in maize.

In eukaryotic organisms, genes typically consist of exons, introns and 5' and 3' untranslated regions (UTRs). Previous studies have shown that gene structure is closely linked to functions and evolution [57]. In the maize PUB gene family, there is a clear difference in the number of exons (Fig. 4). Further analysis revealed that all proteins containing Motif 6 also had Motif 1, suggesting an association between Motif 1 and Motif 6. The majority of U-box proteins contain Motif 1, 2, and 3, with approximately 94% (80) containing Motif 1, 89% (76) containing Motif 2, and 83.5% (71) containing Motif 3, suggesting that these three motifs play an important role in the U-box gene family. The ZmPUB proteins had similar motifs in the common subfamily, while they mostly varied among different subfamilies. It is possible that there is functional redundancy within the same subfamily [58]. Analysis of gene structure revealed that there was considerable variation in gene structure among ZmPUB genes (Table S9). Many ZmPUB genes have similar exon lengths, permutation orders and different intron lengths, which suggests that these genes may have undergone a degree of conservation during the process of evolution. Intron lengths can impact gene expression, alternative splicing, and regulatory processes. The similarity in exon lengths and arrangement suggests shared requirements for encoding core protein information, while the variation in intron lengths may signify regulatory differences and contribute to functional diversity.

Analyzing the gene structure provides further insights, revealing variations in the number of exons and introns among ZmPUB genes. In this study, the number of exons and introns varied in each subfamily (Fig. 4). Analysis of the number of exons and introns can provide more information about the evolution of exon–intron structures and the underlying mechanisms behind different biochemical functions. ZmPUB genes from the same group exhibited similar gene structures, implying functional conservation within the maize PUB gene family. Additionally, variations in exon numbers among ZmPUB genes suggest functional diversity within the maize PUB gene family. The conservation analysis of the gene motifs showed that the PUB genes retained the U-box domain and accumulated additional motifs over the course of evolution. The presence of similar motifs among ZmPUB genes suggests conserved evolutionary relationships and similar biological functions. Exons carry core information required for protein synthesis, while introns protect coding proteins from random harmful mutations [59]. In the PUB gene family, several intronless genes have been reported in multiple species, such as cabbage [24], tomato [25] and Medicago truncatula [60], suggesting the strong structural integrity of PUB genes. These analyses are of great value to scientists interested in the evolution, structure and function of the PUB gene family. Further analysis of the number of exons and introns can provide more information about the evolution of exon–intron structures and the underlying mechanisms behind different biochemical functions.

Exploring the cis-acting elements in ZmPUB genes contributes to revealing their roles in stress response and hormone regulation. We identified 12 major cis-acting elements in the ZmPUB genes (Fig. 5). This suggests that the maize U-box gene family is actively involved in plant development and responses under stress conditions and that the expression of the ZmPUB gene may be stress-regulated and hormonally induced and regulated. In the phytohormone response group, 72% of elements were ABREs, which made up the largest number of elements. ABRE can transcribe and regulate abscisic acid response genes [61], while ABRE_ERD1 has been shown to be upregulated in response to both water stress and etiolation in Arabidopsis, suggesting the essential role of ABRE_ERD1 in dehydration induction and stress response [62]. The PUB gene contains diverse types and quantities of cis-acting elements, suggesting the specificity of the maize PUB gene family in various biological processes. It seems that the presence of these elements implied that ZmPUB genes could be transcriptionally regulated by abiotic stresses.

In our research, the GO annotation and KEGG pathway enrichment analysis provided valuable insights into the diverse roles of ZmPUB proteins, uncovering their involvement in a wide range of biological processes, cellular components, and molecular functions (Fig. 6A). Our findings unveiled the association of ZmPUB proteins with responses to chitin and organonitrogen compounds, indicating their potential role in orchestrating responses to environmental stimuli. Moreover, the results of the KEGG pathway analysis emphasized the significant contribution of U-box genes to various pathways, including those associated with essential cellular processes such as ubiquitin-mediated proteolysis, protein processing in the endoplasmic reticulum, the spliceosome, as well as pathways related to conditions such as Cushing syndrome (Fig. 6B). The significant representation of U-box genes in the ubiquitin-mediated proteolysis pathway further underscores their pivotal role in regulating protein degradation and maintaining cellular homeostasis. The outcomes of the GO annotation and KEGG pathway enrichment analyses portray the maize U-box gene family as multifaceted and pivotal in various biological processes, stressing its involvement in stress responses, hormone regulation, and other cellular processes. These insights align with the comprehensive functional significance exhibited by the diverse cis-acting elements within ZmPUB genes, underlining their potential regulatory role in stress responses and hormone regulation, and reaffirming the coordination between GO and KEGG analyses with the cis-acting elements' analysis.

The tissue expression profiles of ZmPUB genes provide a nuanced understanding of their roles during different developmental processes. Here, we identified expression profiles of maize PUB genes during different tissue development processes (Table S13). The tissue expression heatmaps showed that some ZmPUB genes were specifically expressed in certain tissues (Fig. 7). Some ZmPUB genes were barely expressed in 22 tissues, such as ZmPUB15, ZmPUB19, ZmPUB27, ZmPUB44, ZmPUB48, ZmPUB53, ZmPUB65, ZmPUB66, ZmPUB67 and ZmPUB74 and may have more specific functions. Gene expression patterns are frequently linked to their functions, and analyzing expression patterns can provide vital insights for studying function.

Studies have shown that U-box genes play a crucial role in tolerance to various abiotic stresses, such as salt, drought and heat. Using high-throughput sequencing data, we analyzed the gene expression levels 2, 12 and 24 h after high-salt stress treatment and found different expression of 16 ZmPUB genes under nonbiological stress treatments (Fig. 8B). The qRT-PCR results for genes responding to abiotic stress were consistent with the transcriptome analysis (Fig. 9). In general, more genes were upregulated than downregulated under drought, salt and cold stress. Among the upregulated genes, ZmPUB13, ZmPUB18, ZmPUB19 and ZmPUB68 were consistently upregulated under all three abiotic stress conditions. In particular, these genes were more strongly correlated between salt and drought stress. The expression of ZmPUB59 was upregulated under drought and salt stress but downregulated under high-temperature stress. Interestingly, we observed notable differences in expression at the three time points during the salt stress response, implying different roles for these genes under salt stress. ZmPUB13 and ZmPUB60 were strongly expressed at 12 h under salt stress but at 24 h under drought and high-temperature stress, suggesting that ZmPUB13 and ZmPUB60 respond faster to salt stress than to drought and high-temperature stress. These results show that maize responds differently to abiotic stress at different time points and under different treatments. Several U-box genes were induced under all three stress conditions, suggesting their important role in abiotic stress responses. Further studies are needed to elucidate the specific functions of these ZmPUB genes.

Through homologous analysis between maize and other species, potential similarities in their responses to various abiotic stresses can be revealed. In rice, ZmPUB80 and OsPUB75 are characterized homologous genes. OsPUB75 encodes a cytoplasmic E3 ubiquitin ligase of the RING type and serves as a crucial negative regulator in response to abiotic stress. Specifically, under salt stress, the transcription of OsPUB75 is inhibited [63]. Notably, ZmPUB80 in maize exhibited a similar expression pattern to OsPUB75, showing notably downregulated under high-salt stress. This suggests the potential involvement of ZmPUB80 in the negative regulation of the abiotic stress response. Furthermore, ZmPUB21 and OsPUB41 showed 100% homology (Fig. 1). ZmPUB21 was notably upregulated under salt stress, akin to the role of OsPUB41 as a key negative regulator in the regulation of drought stress tolerance [11]. Similarly, ZmPUB22 and OsPUB15 are homologous genes. OsPUB15 is a negative regulator of cell death and plant response to abiotic stress, conferring high tolerance to salt [10]. Additionally, ZmPUB7, 11, 41 and OsPUB2, 3 are homologous genes, and the homologous U-box E3 ubiquitin ligases OsPUB2 and OsPUB3 are involved in the positive regulation of the response to low-temperature stress [64]. Studies have shown that OsPUB3 acts as a positive regulator of cold stress, and compared to that in wild-type plants, its overexpression in transgenic plants enhances cold stress tolerance [64]. ZmPUB41 was upregulated after 24 h of salt stress treatment. Given its high homology with OsPUB2/3, it is inferred that ZmPUB41 may be involved in the positive regulation of abiotic stress. Moreover, OsPUB45 is homologous to ZmPUB59. OsPUB45 is notably upregulated under drought, salt and cold stress in rice [65], mirrors the expression pattern of ZmPUB59, which exhibited a 4.8-fold upregulation after 2 h of salt stress treatment. The similar expression patterns of ZmPUB59 and OsPUB45 suggest that these two genes may have similar functions. In Arabidopsis, AtPUB18 and AtPUB19 are positively regulated by ABA and salt, and compared to wild-type plants, their double mutants were less sensitive to ABA and salt inhibition of germination [66]. Notably, under salt stress treatment, ZmPUB79 was upregulated by 6.61-fold and showed homology with AtPUB18 and AtPUB19, suggesting that it may be involved in the positive regulation of the salt stress response. A comprehensive analysis of the maize response to abiotic stress is beneficial for selecting specific U-box genes for functional research, enhancing maize stress resistance and breeding superior maize varieties and is important for increasing maize yields.

Our investigation into the subcellular localization of ZmPUB19/59 proteins in the nucleus adds a spatial dimension to their biological functions. The subcellular localization experiment showed that ZmPUB19/59 proteins are located in the nucleus, suggesting that ZmPUB19/59 may exert biological functions in the nucleus (Fig. 10). The high expression levels of ZmPUB19 and ZmPUB59 suggest that they play a crucial role in the abiotic stress response, with the upregulation level of ZmPUB19 reaching tenfold and ZmPUB59 reaching 80-fold after 12 h of salt stress treatment. After 24 h of salt stress treatment, ZmPUB59 was upregulated 120-fold. ZmPUB59 is homologous to OsPUB45. In rice, OsPUB45 is considered a positive regulator of plant defense and stress signaling responses [67], suggesting the important role of ZmPUB59 in the response to abiotic stress. However, the cellular mechanisms of ZmPUB59 in the response to abiotic stress are still unclear and need to be further explored in future studies.

Conclusions

We conducted a systematic bioinformatic analysis of the maize U-box gene family, including analyses of gene structure, conserved motifs, chromosome localization, cis-acting elements, phylogenetic evolution, gene expression and subcellular localization. We predicted the key role of the U-box gene family in maize plant development and response to stress and identified key genes associated with abiotic stress. We analyzed the gene expression of maize under salt, drought and high-temperature stresses, revealing tissue-specific patterns for U-box genes. The conserved U-box genes likely play a critical role in maize adaptation to stress, and multiple U-box genes may cooperate to protect plants from abiotic stress. Our results provide a basis for further analysis of ZmPUB genes to determine their function and elucidate the molecular mechanisms underlying the response of maize to abiotic stress.

Methods

Identification of ZmPUB gene family members in maize

The whole-genome sequences and protein sequences of B73 maize were obtained from MaizeGDB (https://curation.maizegdb.org/) and the Phytozome database (https://phytozome-next.jgi.doe.gov/). The HMM information of the U-box domain (PF04564) was obtained from the Pfam database (https://www.ebi.ac.uk/interpro/entry/pfam/), and the candidate protein sequence containing the U-box domain was selected using HMMER 3.0 (http://hmmer.janelia.org/). The domain information of the candidate protein was analyzed to identify proteins containing U-box domains as ZmPUB proteins in maize using Pfam with an E-value cutoff level of 1.0. The domain was visualized based on the ZmPUB family protein domain from the Pfam database. The UniProt database (https://www.uniprot.org) was used to locate information related to the ZmPUB proteins identified, including domain type, number of amino acids, isoelectric points, relative molecular weight, chromosome position and more.

Analysis of chromosome localization, gene structure, collinearity and cis-acting elements in the promoter region of maize

The chromosomal positions and gene structure of the U-box genes were obtained from the MaizeGDB and Phytozome databases. The similarity of each gene sequence was compared using ClustalOmega (https://www.ebi.ac.uk/Tools/msa/clustalo/). The chromosome location mapping was completed by MapChart software [68]. To assess gene duplication, a BLAST sequence comparison of the U-box genes in maize, Arabidopsis and rice was performed using an e-value < 1e−10 (lower confidence is higher) as a cutoff value. The exon/intron structure of the ZmPUB genes was determined by comparing CDS with genomic sequences using TBtools software for visualization [69]. MCScanX was used to assess repeated events and analyze collinear associations between species (parameters: match score: 50; match size: 5; gap penalty: -1; overlap window: 5; E-value threshold: 10–5; max gaps: 25) [70]. The 1500 bp DNA upstream sequence of the transcription start site of the U-box family gene sequence was downloaded from MaizeGDB. The cis-acting elements were analyzed using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and TBtools software was used to display [69].

Analysis of motif, phylogenesis and tissue-specific expression of ZmPUB gene family members

The U-box protein sequences of maize, rice and Arabidopsis were obtained from the UniProt database. The conserved motifs of ZmPUBs were obtained from MEME (https://meme-suite.org/meme/). The MEME results were displayed with TBtools software [69]. The sequences of the U-box proteins of the three plants were aligned by ClustalX (http://www.clustal.org/), and then the phylogenetic trees of maize, rice and Arabidopsis were constructed by MEGA7 software (Neighbor-Joining, NJ) with the bootstrap parameter set to 1000. RNA-seq data on maize U-box gene expression in different tissues were obtained from MzizeGDB. The 22 tissues spanning the vegetative and reproductive stages of maize development were analyzed to generate a comprehensive and integrated gene expression profile [71]. The results were displayed using R software, where ZmPUB genes were ordered based on a hierarchical clustering analysis.

Gene Ontology (GO) annotation and KEGG pathway enrichment of the maize U-box gene

Proteomic data for maize were obtained from the MaizeGDB database. Functional annotation was performed using eggnog-mapper (http://eggnog-mapper.embl.de) [72]. To better understand the functions of U-box genes, we performed analysis of GO terms and KEGG pathways with the R package clusterProfiler and plotted them by using the ggplot2 package in R [73,74,75]. P values < 0.05 were considered statically significant [76].

Plant materials and stress treatments

Seeds of maize (Zea mays L. inbred strain B73) were sterilized with 3% NaClO for 20 min and washed three times in sterilized water. Then, the seeds were incubated in saturated CaSO4 solution for 6 h. After soaking, the seeds were inoculated onto sterilized filter paper and were kept moist at 26 °C to germinate in the dark for 48 h. Seedlings at the one-leaf stage were transplanted into point-hole plastic plates under the following conditions: 28℃, 1/2-strength Hoagland nutrient solution and a photoperiod of 12/12 h (day/night). The nutrient solution was returned to normal strength after two days. Seedlings at the four-leaf stage were subjected to three abiotic stress treatments. Salt treatment was initiated by nutrient solution containing 300 mM NaCl. Drought treatment was initiated by moving the plants to dry filter paper. High-temperature treatment was initiated at 38℃. After treatment for 0, 2, 12 and 24 h, the seedlings were harvested and stored in liquid nitrogen and then stored in the refrigerator at -80 ℃ for subsequent experiments. The total RNA samples were sent to Genergy Biotechnology (Shanghai) for sequencing on an Illumina® Hiseq3000. Each treated sample consisted of three seedlings. Subsequently, the raw data were processed to eliminate adapter sequences and low-quality reads using Skewer software (v0.2.2) [77]. The resulting clean reads were aligned to the reference genome sequence of maize using STAR software (v2.5.3a) [78].

Quantitative RT-PCR analysis

Total RNA from the stored plants was extracted with TRIzol, and cDNA was obtained using the BeyoRT™ II cDNA kit (Beyotime, Shanghai, China). Quantitative analysis was performed by real-time PCR in conjunction with Hieff® SYBR Green Master Mix (Yeasen) on a LightCycler 480 System (Roche, Vienna, Austria) according to the manufacturer’s instructions. The primers used are listed in Table S15. The following reaction conditions were applied: predenaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and 65 °C for 60 s, and the melting curve was evaluated from 65 °C to 95 °C. The relative transcript levels of the candidate genes were calculated according to the 2−ΔΔCT method. The melting peaks of the candidate genes are displayed in Fig. S3.

Construction of vectors and subcellular localization

The subcellular localization of the U-box family genes was predicted using Plant-PLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant) by uploading protein sequences. Two U-box genes, ZmPUB19 and ZmPUB59, were cloned using In-Fusion technology. Primers were designed using online software, and sequence information can be found in Additional file 4: Table S15. The PCR products of ZmPUB19 and ZmPUB59 were linked to the 5’ end of the mGFP of the pGreenII-35S-mGFP cloning vector and then transformed into DH5α. The resulting fusion expression vectors, pGreenII-ZmPUB19 and pGreenII-ZmPUB59, were validated by sequencing. Subsequently, the validated fusion expression vectors were transformed into Agrobacterium strain GV3101 (pSoup). The nucleus marker was created by linking the NLS sequence to pGreenII-35S-mCherry and was tested with infected Nicotiana leaf cells [79]. The constructed vector plasmid was transformed into Agrobacterium strain GV3101 (pSoup), and the vectors were transiently transformed into Nicotiana leaves [80]. The infected tobacco plants were cultured in the dark at 25 °C for 12 h and then returned to normal culture conditions for 24 h. Finally, the infected cells of the lower epidermis of the transformed leaves were imaged using a confocal laser microscope (OLYMPUS FV3000, Japan) at an excitation wavelength of 488 nm to detect GFP and at an excitation wavelength of 561 nm to detect mCherry.

Statistical analysis

In this study, the qRT-PCR results are reported as the means of three independent experiments. The significant differences between treatments were analyzed using standard deviation and one-way analysis of variance (ANOVA). The significant differences between the control group and the treatment groups were assessed by Student’s t test. The result of P < 0.05 was used as the significance threshold (* P < 0.05, ** P < 0.01 and *** P < 0.001).

Availability of data and materials

The datasets generated during the current study are available in the GenBank repository (http://www.ncbi.nlm.nih.gov/Genbank) and the MaizeGDB (http://www.maizegdb.org), and their public access to these databases is open. All data generated or analyzed during this study are included in this article and its supplementary information files.

Abbreviations

E3:

E3 ubiquitin ligase enzyme

NJ:

Neighbor-joining

PUB:

Plant U-box

ZmPUB:

Maize PUB

References

  1. Kajla M, Yadav VK, Khokhar J, Singh S, Chhokar RS, Meena RP, Sharma RK. Increase in wheat production through management of abiotic stresses: a review. J Appl Nat Sci. 2015;7(2):1070–80.

    CAS  Google Scholar 

  2. Wang S, Lv X, Zhang J, Chen D, Chen S, Fan G, Ma C, Wang Y. Roles of E3 ubiquitin ligases in plant responses to abiotic stresses. INT J Mol Sci. 2022;23(4):2308.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Aravind L, Koonin EV. The U box is a modified RING finger - a common domain in ubiquitination. Curr Biol. 2000;10(4):R132-134.

    Article  CAS  PubMed  Google Scholar 

  4. Pickart CM. Mechanisms underlying ubiquitination. Annu Rev Biochem. 2001;70:503–33.

    Article  CAS  PubMed  Google Scholar 

  5. Finley D, Chau V. Ubiquitination. Annu Rev Cell Biol. 1991;7:25–69.

    Article  CAS  PubMed  Google Scholar 

  6. Mazzucotelli E, Belloni S, Marone D, De Leonardis A, Guerra D, Di Fonzo N, Cattivelli L, Mastrangelo A. The e3 ubiquitin ligase gene family in plants: regulation by degradation. Curr Genomics. 2006;7(8):509–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Duplan V, Rivas S. E3 ubiquitin-ligases and their target proteins during the regulation of plant innate immunity. Front Plant Sci. 2014;5:42.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Moon J, Parry G, Estelle M. The ubiquitin-proteasome pathway and plant development. Plant Cell. 2004;16(12):3181–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Koegl M, Hoppe T, Schlenker S, Ulrich HD, Mayer TU, Jentsch S. A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell. 1999;96(5):635–44.

    Article  CAS  PubMed  Google Scholar 

  10. Park JJ, Yi J, Yoon J, Cho LH, Ping J, Jeong HJ, Cho SK, Kim WT, An G. OsPUB15, an E3 ubiquitin ligase, functions to reduce cellular oxidative stress during seedling establishment. Plant J. 2011;65(2):194–205.

    Article  CAS  PubMed  Google Scholar 

  11. Seo DH, Lee A, Yu SG, Cui LH, Min HJ, Lee SE, Cho NH, Kim S, Bae H, Kim WT. OsPUB41, a U-box E3 ubiquitin ligase, acts as a negative regulator of drought stress response in rice (Oryza Sativa L.). Plant Mol Biol. 2021;106(4–5):463–77.

    Article  CAS  PubMed  Google Scholar 

  12. Wang W, Wang W, Wu Y, Li Q, Zhang G, Shi R, Yang J, Wang Y, Wang W. The involvement of wheat U-box E3 ubiquitin ligase TaPUB1 in salt stress tolerance. J Integr Plant Biol. 2020;62(5):631–51.

    Article  CAS  PubMed  Google Scholar 

  13. Li Q, Li B, Wang J, Chang X, Mao X, Jing R. TaPUB15, a U-Box E3 ubiquitin ligase gene from wheat, enhances salt tolerance in rice. Food Energy Secur. 2021;10(1):15.

  14. Wu Y, Wang W, Li Q, Zhang G, Zhao X, Li G, Li Y, Wang Y, Wang W. The wheat E3 ligase TaPUB26 is a negative regulator in response to salt stress in transgenic Brachypodium distachyon. Plant Sci. 2020;294: 110441.

    Article  CAS  PubMed  Google Scholar 

  15. Yang L, Wu L, Chang W, Li Z, Miao M, Li Y, Yang J, Liu Z, Tan J. Overexpression of the maize E3 ubiquitin ligase gene ZmAIRP4 enhances drought stress tolerance in Arabidopsis. Plant Physiol Bioch. 2018;123:34–42.

    Article  CAS  Google Scholar 

  16. Xia Z, Liu Q, Wu J, Ding J. ZmRFP1, the putative ortholog of SDIR1, encodes a RING-H2 E3 ubiquitin ligase and responds to drought stress in an ABA-dependent manner in maize. Gene. 2012;495(2):146–53.

    Article  CAS  PubMed  Google Scholar 

  17. Wang N, Liu Y, Cai Y, Tang J, Li Y, Gai J. The soybean U-box gene GmPUB6 regulates drought tolerance in Arabidopsis thaliana. Plant Physiol Bioch. 2020;155:284–96.

    Article  CAS  Google Scholar 

  18. Qi S, Lin Q, Zhu H, Gao F, Zhang W, Hua X. The RING finger E3 ligase SpRing is a positive regulator of salt stress signaling in salt-tolerant wild tomato species. Plant Cell Physiol. 2016;57(3):528–39.

    Article  CAS  PubMed  Google Scholar 

  19. Tang X, Ghimire S, Liu W, Fu X, Zhang H, Zhang N, Si H. Potato E3 ubiquitin ligase PUB27 negatively regulates drought tolerance by mediating stomatal movement. Plant Physiol Bioch. 2020;154:557–63.

    Article  CAS  Google Scholar 

  20. Lim CW, Baek W, Lee SC. The Pepper RING-Type E3 ligase CaAIRF1 regulates ABA and drought signaling via CaADIP1 protein phosphatase degradation. Plant Physiol. 2017;173(4):2323–39.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. An J, Liu X, Song L, You C, Wang X, Hao Y. Apple RING finger E3 ubiquitin ligase MdMIEL1 negatively regulates salt and oxidative stresses tolerance. J Plant Biol. 2017;60(2):137–45.

    Article  CAS  Google Scholar 

  22. Liu YC, Wu YR, Huang XH, Sun J, Xie Q. AtPUB19, a U-box E3 ubiquitin ligase, negatively regulates abscisic acid and drought responses in Arabidopsis thaliana. Mol Plant. 2011;4(6):938–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zeng LR, Park CH, Venu RC, Gough J, Wang GL. Classification, expression pattern, and E3 ligase activity assay of rice u-box-containing proteins. Mol Plant. 2008;1(5):800–15.

    Article  CAS  PubMed  Google Scholar 

  24. Wang C, Duan W, Riquicho AR, Jing Z, Liu T, Hou X, Li Y. Genome-wide survey and expression analysis of the PUB family in Chinese cabbage (Brassica rapa ssp. pekinesis). Mol Genet Genomics. 2015;290(6):2241–60.

    Article  CAS  PubMed  Google Scholar 

  25. Mandal A, Mishra AK, Dulani P, Muthamilarasan M, Shweta S, Prasad M. Identification, characterization, expression profiling, and virus-induced gene silencing of armadillo repeat-containing proteins in tomato suggest their involvement in tomato leaf curl New Delhi virus resistance. Funct Integr Genomic. 2018;18(2):101–11.

    Article  CAS  Google Scholar 

  26. Ryu MY, Cho SK, Hong Y, Kim J, Kim JH, Kim GM, Chen YJ, Knoch E, Moller BL, Kim WT, et al. Classification of barley U-box E3 ligases and their expression patterns in response to drought and pathogen stresses. BMC Genomics. 2019;20(1):326.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Lu X, Shu N, Wang D, Wang J, Chen X, Zhang B, Wang S, Guo L, Chen C, Ye W. Genome-wide identification and expression analysis of PUB genes in cotton. BMC Genomics. 2020;21(1):213.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kim DY, Lee YJ, Hong MJ, Kim JH, Seo YW. Genome wide analysis of U-Box E3 Ubiquitin Ligases in Wheat (Triticum aestivum L). Int J Mol Sci. 2021;22(5):2699.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Fang Y, Du Q, Yang Q, Jiang J, Hou X, Yang Z, Zhao D, Li X, Xie X. Identification, characterization, and expression profiling of the putative U-box E3 ubiquitin ligase gene family in Sorghum bicolor. Front Microbiol. 2022;13: 942302.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Tang X, Ghimire S, Liu W, Fu X, Zhang H, Sun F, Zhang N, Si H. Genome-wide identification of U-box genes and protein ubiquitination under PEG-induced drought stress in potato. Physiol Plant. 2022;174(1):e13475.

    Article  CAS  PubMed  Google Scholar 

  31. Wang P, Zhu L, Li Z, Cheng M, Chen X, Wang A, Wang C, Zhang X. Genome-wide identification of the U-Box E3 ubiquitin ligase Gene Family in Cabbage (Brassica oleracea var. capitata) and its expression analysis in response to cold stress and Pathogen infection. Plants. 2023;12(7):1437.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hu D, Xie Q, Liu Q, Zuo T, Zhang H, Zhang Y, Lian X, Zhu L. Genome-wide distribution, expression and function analysis of the U-Box Gene Family in Brassica oleracea L. Genes-Basel. 2019;10(12):1000.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Yang G, Ying G, Wang Z, Pan W, Linghu B, Pan Y, Song W, Cui L, Nie X. Genome-wide identification and expression analysis of U-box gene family in wild emmer wheat (Triticum turgidum L. ssp. dicoccoides). Gene. 2021;799:145840.

    Article  CAS  PubMed  Google Scholar 

  34. Hajibarat Z, Saidi A, Zeinalabedini M, Gorji AM, Ghaffari MR, Shariati V, Ahmadvand R. Genome-wide identification of StU-box gene family and assessment of their expression in developmental stages of Solanum tuberosum. J Genet Eng Biotechnol. 2022;20(1):25.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Hu H, Dong C, Sun D, Hu Y, Xie J. Genome-Wide Identification and Analysis of U-Box E3 Ubiquitin(-)Protein Ligase Gene Family in Banana. Int J Mol Sci. 2018;19(12):3874.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Tan B, Lian X, Cheng J, Zeng W, Zheng X, Wang W, Ye X, Li J, Li Z, Zhang L, et al. Genome-wide identification and transcriptome profiling reveal that E3 ubiquitin ligase genes relevant to ethylene, auxin and abscisic acid are differentially expressed in the fruits of melting flesh and stony hard peach varieties. BMC Genomics. 2019;20(1):892.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wang C, Song B, Dai Y, Zhang S, Huang X. Genome-wide identification and functional analysis of U-box E3 ubiquitin ligases gene family related to drought stress response in Chinese white pear (Pyrus Bretschneideri). BMC Plant Biol. 2021;21(1):235.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Wang K, Yang Q, Lanhuang B, Lin H, Shi Y, Dhanasekaran S, Godana EA, Zhang H. Genome-wide investigation and analysis of U-box Ubiquitin–Protein ligase gene family in apple: expression profiles during Penicillium expansum infection process. Physiol Mol Plant P. 2020;111:101487.

    Article  CAS  Google Scholar 

  39. Jiang L, Lin Y, Wang L, Peng Y, Yang M, Jiang Y, Hou G, Liu X, Li M, Zhang Y, et al. Genome-wide identification and expression profiling reveal the regulatory role of U-box E3 ubiquitin ligase genes in strawberry fruit ripening and abiotic stresses resistance. Front Plant Sci. 2023;14: 1171056.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Huber AH, Weis WI. The structure of the beta-catenin/E-cadherin complex and the molecular basis of diverse ligand recognition by beta-catenin. Cell. 2001;105(3):391–402.

    Article  CAS  PubMed  Google Scholar 

  41. Amador V, Monte E, Garcia-Martinez JL, Prat S. Gibberellins signal nuclear import of PHOR1, a photoperiod-responsive protein with homology to Drosophila armadillo. Cell. 2001;106(3):343–54.

    Article  CAS  PubMed  Google Scholar 

  42. Yang X, Kalluri UC, Jawdy S, Gunter LE, Yin T, Tschaplinski TJ, Weston DJ, Ranjan P, Tuskan GA. The F-box gene family is expanded in herbaceous annual plants relative to woody perennial plants. Plant Physiol. 2008;148(3):1189–200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Zhang Z. KaKs_Calculator 3.0: calculating selective pressure on Coding and non-coding sequences.  Genomics Proteomics Bioinformatics. 2022;20(3):536–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Gong F, Yang L, Tai F, Hu X, Wang W. “Omics” of maize stress response for sustainable food production: opportunities and challenges. OMICS. 2014;18(12):714–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Li X, Zhu L, Wu Z, Chen J, Wang T, Zhang X, Mei G, Wang J, Lv G. Classification and Expression Profile of the U-Box E3 Ubiquitin Ligase Enzyme Gene Family in Maize (Zea mays L.). Plants-Basel. 2022;11(19):2459.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Peifer M, Berg S, Reynolds AB. A repeating amino acid motif shared by proteins with diverse cellular roles. Cell. 1994;76(5):789–91.

    Article  CAS  PubMed  Google Scholar 

  47. Groves MR, Barford D. Topological characteristics of helical repeat proteins. Curr Opin Struc Biol. 1999;9(3):383–9.

    Article  CAS  Google Scholar 

  48. Patterson C. A new gun in town: the U box is a ubiquitin ligase domain. Sci STKE. 2002;2002(116): e4.

    Article  Google Scholar 

  49. Mudgil Y, Shiu SH, Stone SL, Salt JN, Goring DR. A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family. Plant Physiol. 2004;134(1):59–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Cho SK, Chung HS, Ryu MY, Park MJ, Lee MM, Bahk YY, Kim J, Pai HS, Kim WT. Heterologous expression and molecular and cellular characterization of CaPUB1 encoding a hot pepper U-Box E3 ubiquitin ligase homolog. Plant Physiol. 2006;142(4):1664–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Yee D, Goring DR. The diversity of plant U-box E3 ubiquitin ligases: from upstream activators to downstream target substrates. J Exp Bot. 2009;60(4):1109–21.

    Article  CAS  PubMed  Google Scholar 

  52. Chang D, Duda TJ. Extensive and continuous duplication facilitates rapid evolution and diversification of gene families. Mol Biol Evol. 2012;29(8):2019–29.

    Article  CAS  PubMed  Google Scholar 

  53. Xiao J, Sekhwal MK, Li P, Ragupathy R, Cloutier S, Wang X, You FM. Pseudogenes and their genome-wide prediction in plants. Int J Mol Sci. 2016;17(12):1991.

    Article  PubMed  PubMed Central  Google Scholar 

  54. Parey E, Louis A, Cabau C, Guiguen Y, Roest CH, Berthelot C. Synteny-guided resolution of Gene Trees clarifies the functional impact of whole-genome duplications. Mol Biol Evol. 2020;37(11):3324–37.

    Article  CAS  PubMed  Google Scholar 

  55. Freeling M. Bias in plant gene content following different sorts of duplication: tandem, whole-genome, segmental, or by transposition. Annu Rev Plant Biol. 2009;60:433–53.

    Article  CAS  PubMed  Google Scholar 

  56. Wagner A. Gene duplications, robustness and evolutionary innovations. BioEssays. 2008;30(4):367–73.

    Article  CAS  PubMed  Google Scholar 

  57. Xu G, Guo C, Shan H, Kong H. Divergence of duplicate genes in exon-intron structure. P Natl Acad Sci USA. 2012;109(4):1187–92.

    Article  CAS  Google Scholar 

  58. Leng X, Wei H, Xu X, Ghuge SA, Jia D, Liu G, Wang Y, Yuan Y. Genome-wide identification and transcript analysis of TCP transcription factors in grapevine. BMC Genomics. 2019;20(1):786.

    Article  PubMed  PubMed Central  Google Scholar 

  59. Jo BS, Choi SS. Introns: the functional benefits of introns in genomes. Genomics Inf. 2015;13(4):112–8.

    Article  Google Scholar 

  60. Song J, Mo X, Yang H, Yue L, Song J, Mo B. The U-box family genes in Medicago truncatula: key elements in response to salt, cold, and drought stresses. PLoS One. 2017;12(8):e182402.

    Article  Google Scholar 

  61. Osakabe Y, Yamaguchi-Shinozaki K, Shinozaki K, Tran LP. ABA control of plant macroelement membrane transport systems in response to water deficit and high salinity. New Phytol. 2014;202(1):35–49.

    Article  PubMed  Google Scholar 

  62. Simpson SD, Nakashima K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. Two different novel cis-acting elements of erd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence. Plant J. 2003;33(2):259–70.

    Article  CAS  PubMed  Google Scholar 

  63. Sharma M, Pandey GK. OsPUB75, an Armadillo/U-box protein interacts with GSK3 kinase and functions as negative regulator of abiotic stress responses. Environ Exp Bot. 2019;161:388–98.

    Article  CAS  Google Scholar 

  64. Byun MY, Cui LH, Oh TK, Jung YJ, Lee A, Park KY, Kang BG, Kim WT. Homologous U-box E3 ubiquitin ligases OsPUB2 and OsPUB3 are involved in the positive regulation of low temperature stress response in Rice (Oryza sativa L). Front Plant Sci. 2017;8:16.

    Article  PubMed  PubMed Central  Google Scholar 

  65. Sharma M, Singh A, Shankar A, Pandey A, Baranwal V, Kapoor S, Tyagi AK, Pandey GK. Comprehensive expression analysis of rice Armadillo gene family during abiotic stress and development. DNA Res. 2014;21(3):267–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Bergler J, Hoth S. Plant U-box armadillo repeat proteins AtPUB18 and AtPUB19 are involved in salt inhibition of germination in Arabidopsis. Plant Biol. 2011;13(5):725–30.

    Article  CAS  PubMed  Google Scholar 

  67. Yang CW, Gonzalez-Lamothe R, Ewan RA, Rowland O, Yoshioka H, Shenton M, Ye H, O’Donnell E, Jones JD, Sadanandom A. The E3 ubiquitin ligase activity of arabidopsis PLANT U-BOX17 and its functional tobacco homolog ACRE276 are required for cell death and defense. Plant Cell. 2006;18(4):1084–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Voorrips RE. MapChart: software for the graphical presentation of linkage maps and QTLs. J Hered. 2002;93(1):77–8.

    Article  CAS  PubMed  Google Scholar 

  69. Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R. TBtools: an integrative Toolkit developed for interactive analyses of big Biological Data. Mol Plant. 2020;13(8):1194–202.

    Article  CAS  PubMed  Google Scholar 

  70. Wang Y, Tang H, Debarry JD, Tan X, Li J, Wang X, Lee TH, Jin H, Marler B, Guo H, et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40(7):e49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Walley JW, Sartor RC, Shen Z, Schmitz RJ, Wu KJ, Urich MA, Nery JR, Smith LG, Schnable JC, Ecker JR, et al. Integration of omic networks in a developmental atlas of maize. Science. 2016;353(6301):814–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Cantalapiedra CP, Hernandez-Plaza A, Letunic I, Bork P, Huerta-Cepas J. eggNOG-mapper v2: functional annotation, Orthology assignments, and Domain Prediction at the Metagenomic Scale. Mol Biol Evol. 2021;38(12):5825–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Kanehisa M. Toward understanding the origin and evolution of cellular organisms. PROTEIN SCI. 2019;28(11):1947–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Kanehisa M, Furumichi M, Sato Y, Kawashima M, Ishiguro-Watanabe M. KEGG for taxonomy-based analysis of pathways and genomes. NUCLEIC ACIDS RES. 2023;51(D1):D587-592.

    Article  CAS  PubMed  Google Scholar 

  76. Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z, Feng T, Zhou L, Tang W, Zhan L, et al. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innov (Camb). 2021;2(3):100141.

    CAS  Google Scholar 

  77. Jiang H, Lei R, Ding SW, Zhu S. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics. 2014;15: 182.

    Article  PubMed  PubMed Central  Google Scholar 

  78. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29(1):15–21.

    Article  CAS  PubMed  Google Scholar 

  79. Wu G, Spalding EP. Separate functions for nuclear and cytoplasmic cryptochrome 1 during photomorphogenesis of Arabidopsis seedlings. P Natl Acad Sci USA. 2007;104(47):18813–8.

    Article  CAS  Google Scholar 

  80. Blatt MR, Grefen C. Applications of fluorescent marker proteins in plant cell biology. Methods Mol Biol. 2014;1062:487–507.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

Special thanks to the reviewers for their precious time and valuable suggestions.

Funding

This study was financed by the National Natural Science Foundation of China (Grant No. 32060069 and No. 32260068) and the Natural Science Foundation of Jiangxi Province (Grant No. 20212ACB215004).

Author information

Author notes

  1. Yongle Liu, Changgen Li and Aokang Qin contributed equally to this work.

Authors and Affiliations

  1. College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang, 330045, People’s Republic of China

    Yongle Liu, Changgen Li, Aokang Qin, Wenli Deng, Rongrong Chen, Hongyang Yu, Yihua Wang, Jianbo Song & Liming Zeng

  2. College of Life Sciences, Nanjing University, Nanjing, 210095, People’s Republic of China

    Yongle Liu

  3. Jiangxi Provincial Key Laboratory of Conservation Biology, Jiangxi Agricultural University, Nanchang, 330045, People’s Republic of China

    Jianbo Song

Authors
  1. Yongle Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Changgen Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aokang Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wenli Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rongrong Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hongyang Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yihua Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jianbo Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Liming Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

YL, CL and AQ carried out data collection. AQ and YL analyzed the data. YL and CL performed the biological experiment and wrote the manuscript. WD prepared the plant sample. JS and LZ contributed to the study conception and design. RC, YW and HY carried out the revisions. All authors have read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Jianbo Song or Liming Zeng.

Ethics declarations

Ethics approval and consent to participate

All plant materials involved in this research are used for scientific research and were allowed to be used and provided free of charge in this study. The Zea mays L. seeds utilized in this study were graciously provided by Professor Li Hua of Henan Agricultural University. These plant materials do not include any wild species at risk of extinction. No specific permits were required for sample collection in this study. We complied with relevant institutional, national, and international guidelines and legislation for plant study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, Y., Li, C., Qin, A. et al. Genome-wide identification and transcriptome profiling expression analysis of the U-box E3 ubiquitin ligase gene family related to abiotic stress in maize (Zea mays L.). BMC Genomics 25, 132 (2024). https://doi.org/10.1186/s12864-024-10040-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12864-024-10040-8

Keywords

BMC Genomics

ISSN: 1471-2164

Contact us