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Genome-wide identification and expression analysis of the U-box gene family related to biotic and abiotic stresses in Coffea canephora L.

BMC Genomics volume 25, Article number: 916 (2024) Cite this article

Abstract

Plant U-box genes play an important role in the regulation of plant hormone signal transduction, stress tolerance, and pathogen resistance; however, their functions in coffee (Coffea canephora L.) remain largely unexplored. In this study, we identified 47 CcPUB genes in the C. canephora L. genome, clustering them into nine groups via phylogenetic tree. The CcPUB genes were unevenly distributed across the 11 chromosomes of C. canephora L., with the majority (11) on chromosome 2 and none on chromosome 8. The cis-acting elements analysis showed that CcPUB genes were involved in abiotic and biotic stresses, phytohormone responsive, and plant growth and development. RNA-seq data revealed diverse expression patterns of CcPUB genes across leaves, stems, and fruits tissues. qRT-PCR analyses under dehydration, low temperature, SA, and Colletotrichum stresses showed significant up-regulation of CcPUB2, CcPUB24, CcPUB34, and CcPUB40 in leaves. Furthermore, subcellular localization showed CcPUB2 and CcPUB34 were located in the plasma membrane and nucleus, and CcPUB24 and CcPUB40 were located in the nucleus. This study provides valuable insights into the roles of PUB genes in stress responses and phytohormone signaling in C. canephora L., and provided basis for functional characterization of PUB genes in C. canephora L.

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Introduction

Plants are frequently exposed to complex environmental conditions including biotic and abiotic stresses in their life [1]. To counter these destructive stresses, plants have formed complex and precise pathways [2], including transcriptional regulation, post-transcriptional modifications, epigenetic regulation, and post-translational modifications [3]. Ubiquitination, a key post-translational modification pathway, involves the ubiquitin/26S proteasome (UPS) system, which includes the E1 ubiquitin (UB) activating enzyme, E2 UB conjugating enzyme, and E3 UB ligase [4, 5]. The UPS system operates through a series of ATP-dependent reactions where an E1 enzyme links ubiquitin to its C-terminal, transferring it to an E2 enzyme. The E2 enzyme then facilitates the attachment of ubiquitin to the target protein in the presence of an E3 ligase, which recognizes and binds to specific degradation signals on target proteins [6, 7]. E3 ligases in plants are categorized into four types: HECT, RING, U-box [3, 8], and SCF [9]. Arabidopsis thaliana has over 1300 E3 ligases, constituting more than 5% of its proteome and regulating nearly 2600 target proteins [10,11,12].

The first U-box ubiquitin ligase was clarified from ubiquitin fusion degradation protein-2 (UFD2) in yeast [13]. Plant U-box (PUB) proteins, characterized by a U-box domain containing approximately 75 amino acids, are involved in various growth and development processes, such as hormone responses, seed germination, and responses to biotic and abiotic stresses [14, 15].

Analyzing the structure and distribution of PUB proteins enhances our understanding of their mechanisms of action [5, 16]. Previous studies have identified and analyzed PUB family genes in higher plants, including A. thaliana (64 genes) [17], Oryza sativa (77 genes) [12], Brassica rapa ssp. pekinesis (101 genes) [18], Glycine max (125 genes) [19], Musa acuminata (91 genes) [20], Brassica oleracea (99 genes) [21], Gossypium hirsutum (185 genes) [22], Solanum lycopersicum (62 genes) [23], Pyrus bretschneideri (62 genes) [24], Phyllostachys edulis (121 genes) [25], Solanum tuberosum (62 genes) [26], and Sorghum bicolor (59 genes) [27]. However, the PUB gene family has been rarely studied in coffee.

Many studies showed that PUB proteins play an important role in the regulation of plant hormone signal transduction, stress tolerance, and pathogen resistance. In A. thaliana, AtPUB9/18/19/44 have involved in the ABA biosynthesis [28, 29], AtPUB17 was required for cell death and defense [30], AtPUB22/23/24 have been shown to play a role in plant resistance to Fusarium oxysporum [31], and overexpression AtPUB46 in A. thaliana enhances tolerance to drought and oxidative stress [32], AtPUB48 play key roles in heat tolerance of Arabidopsis [33]. In rice, OsPUB15 mutation seeds did not produce primary roots, and their shoot development was significantly delayed [34]. And overexpressing OsPUB15 transgenic rice enhanced resistance to blast strains [35]. OsPUB24 play an important role in regulating brassinosteroid (BR) response in rice [36]. Wheat TaPUB1 and TaPUB26 have been proved to regulate salt stress tolerance in Triticum aestivum [37, 38]. In G. max, GmPUB8 was shown to negatively regulate to salt and drought stress in seed germination and post-germination stages [19]. Heterogeneous overexpression of PnSAG1 in Physcomitrella patens and Arabidopsis enhanced the sensitivity to the salinity and ABA [39]. In Malus domestica Borkh., MdPUB29 may positively regulate salt tolerance [40].

Coffee (Coffea arabica L. and C. canephora L.) is a major tropical beverage crop native to the highlands of southwest Ethiopia [41]. Introduced to China in the late 19th century, both Robusta and Arabica species are now widely cultivated in Hainan and Yunnan provinces, respectively, becoming economically valuable crops [42]. Despite extensive research on PUB genes in other plants, their roles in coffee remain unclear. This study aims to identify and analyze PUB genes in C. canephora L., examining their gene structure and evolution, expression patterns in different tissues, and response to various stresses. Our findings provide new insights into the functions of PUB genes in coffee.

Materials and methods

The data retrieval and identification of plant U-box proteins

The genomes annotation of 7 species (A. thaliana, C. canephora, O. sativa, Brachypodium distachyon, Amborella trichopoda, Theobroma cacao, Vitis vinifera) was chosen in this study to identify U-box proteins (Table S1). The Hidden Markov Model (HMM) file for the U-box domain (PF04564) was retrieved from the Pfam website (https://pfam.xfam.org/) to search U-box proteins by performing the Hmmsearch program in Hmmer software (version 3.2) [43, 44]. Next, domains of the protein sequences were further identified by Interproscan to confirm the U-box domain (SM00185 in the SMART database and PF04564 in the PFAM database [45]. The protein properties of U-box proteins were predicted by the ProtParam module in Biopython [46].

Gene structure, chromosomal distribution, conserved motifs and phylogenetic analysis of CcPUBs

The information on exon/intron position in the C. canephora L. was retrieved from the corresponding genomic annotation files (GFF/GTF files). The protein sequences were submitted to the MEME software [47]. The TBtools (version 1.0987663) software was used to visualize the gene structure and protein motifs [48]. The phylogenetic trees were constructed by IQTREE using the maximum likelihood (ML) method with 1000 bootstrap replications [49].

Identification of gene collinearity and specific duplication events

The collinearity gene pairs of C. canephora L. were identified by BlastP (E < 1 × 10 − 10, top 5 matches, and m8 format output) and MCScanX (with default parameter) [50]. The collinearity genes pairs and the chromosome location were visualized by Circos (version 0.69-9) [51]. The duplication event and the related gene pairs within C. canephora L. was identified and classified by MCScanX and Dupgen_finder [52].

Analysis of cis-elements in the promoter of CcPUBs

The cis-elements in the 2000 bp upstream genomic DNA sequences were submitted to the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) to predict the cis-elements [53].

RNA-seq analysis

Two sets of transcriptome data from the previous studies were chosen in this study, including PRJNA561881 (coffee bean of 10 years old Robusta under normal) and PRJNA798825 (leaf and stem of 10 years old Robusta under normal).

The FastQC (v0.11.9) and trimmomatic (v0.3.9) were performed to evaluate the sequence quality and filter the low-quality reads, respectively. The HISAT2 (version 2.1.0) was employed to build the index file. By performing the HISAT and samtools, the clean reads were mapped to the reference genome and transformed to the BAM format data. The transcripts were assembled by Stringtie (v2.0) software, and the TPM (Transcripts Per Kilobase Million) value was generated by the R software. The genes with | logFC | > 1 and p-value 0.05 were chosen as DEGs and identified by edgeR.

Plant and fungi material

Colletotrichum isolates strain C. fructicola (stored in our lab), C. canephora L. varieties Reyan 1 cultivated by Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences, and Nicotiana benthamiana were used in this study. The fungus was cultured in liquid Czapek Dox medium or on potato dextrose agar medium (PDA) for 5 d at 25 °C. After sowing, C. canephora L. plants and N. benthamiana was grown in a greenhouse at 23 °C (dark)/28°C (light) with a 16 h light/8 h dark photoperiod.

Stress treatments

To further explore the function of CcPUBs during stresses, the seedlings were subjected to four different abiotic and biotic stresses, including dehydration, low temperature, salicylic acid (SA) and Colletotrichum treatment. The method of abiotic treatment was performed as previous study [54, 55]. Seven time points (0, 1, 3, 6, 9, 12 and 24 h) were selected to collected leaves of seeding under stresses. In SA treatment, seven time points were selected (0, 1, 3, 6, 9, 12 and 24 h). The method of biotic treatment was performed as previous study [56]. Detached and intact leaves of 6-month coffee seedlings were inoculated with Φ5mm mycelial discs of the isolates. Seven time points (0, 1, 3, 6, 9, 12 and 24 h) were selected to collected leaves of seeding under C. fructicola stress.

Subcellular localization of seclected CcPUB members

According to the previous method [57], the full-length coding sequences of the selected CcPUB genes (CcPUB2, CcPUB24, CcPUB34 and CcPUB40) were amplified from cDNA of C. canephora L. levaes using specific primers. And all sequences were integrated into the pCAMBIA2300-YFP vector. The CcPUBs-YFP constructs were transformed into Agrobacterium tumefaciens strain GV3101. The positive cell cultures of CcPUBs-YFP were infiltrated into N. benthamiana leaves. The YFP fluorescence signals were observed using a confocal microscope at 488 nm. The well-characterized PUB members GhARM144-GFP and OsPUB41-GFP fluorescence signals were observed using a confocal microscope at 488 nm. The mCherry fluorescence signals were observed using a confocal microscope at 594 nm. Each assay was performed on three leaves from three individual plants and repeated three times. The primers used are listed in Table S2.

Nucleic acid extraction and RT-qPCR analysis

Total RNA was isolated using FastPure® Plant RNA Isolation Mini Kit (Vazyme, RC411-C1), quantified and used as a template for reverse transcription with the HiScript® II Q RT SuperMix for qPCR (+ g DNA wiper) (Vazyme, R223-1). The RT-qPCR assays were performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02). The Tubulin gene was used as internal control [58]. The experiment was repeated three times. The sequences of each of the primers used in the different RT-qPCR assays are listed in Table S2.

Results

Identification of CcPUB gene family member

In this study, 47 CcPUB genes were identified in the reference genome of C. canephora L.(A2, 2n = 2x = 12)by detecting the U-box conserved domain via Pfam (PF04564). The chromosome location, isoelectric point (pI), amino acid length, and molecular weight (MW) of these genes was analyzed and listed in Table S3. The 47 CcPUB proteins ranged from 127 (CcPUB24) to 1488 (CcPUB41) amino acids (aa) in length. The molecular weight varied from 14.44 to 165.62 kDa. The theoretical isoelectric point (pI) ranged from 5.04 (CcPUB24) to 8.99 (CcPUB36). The grand average of hydropathy (GRAVY) ranged from negative values to positive values (-0.772 ~ 0.206), indicating that both of the hydrophilic hydrophobic proteins existed in CcPUB proteins (Table S3). CcPUB genes were unevenly distributed on every chromosome (Table S3). For C. canephora L. genome, the PUB genes were mainly distributed on chromosomes 01, 02, and 07.

Phylogenetic analysis of CcPUB genes

To investigate the evolutionary history of PUB genes in C. canephora L. Together with the AtPUB (61 members), OsPUB (77 members), TcPUB (56 members), BdPUB (63 members), VvPUB (49 members), AmbPUB (37 members), and 47 CcPUB proteins, a phylogenetic tree including 390 PUB proteins was generated. As shown in Fig. 1, these PUB proteins can be divided into eleven distinct subfamilies, namely Group A to K, no species-specific subfamily was found. The members in Group E constituted the largest number (95 members), whereas the Group B had the lowest number (5 members) (Fig. 1). Groups E and F were the largest with 11 CcPUB members, followed by the Groups D, I and F with 5 CcPUB members, respectively, while Groups B and C had no CcPUB members (Fig. 1). In addition, monocots species PUB members were more than dicots species PUBs. For example, there were 77 PUB members in O. sativa, and there were 47 PUBs in coffee. It indicated that lineage-specific expansion events also occurred in higher land monocots (O. sativa), and extra PUB members were generated after the divergence from dicots (C. canephora L.).

Fig. 1
figure 1

The phylogenetic tree of plant U-boxs (PUB) family. The maximum likelihood (ML) tree was construct by the full-length amino acid sequences of PUB genes from C. canephora L. (Cc), O. sativa (Os), B. distachyon (Bd), A. trichopoda (Amb), A. thaliana (At), T. cacao (Ta), and V. vinifera (Vv), using MEGA X, with 1000 replicates. They were divided into three groups. Group-A to Group-K was represented by different colour, respectively

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Conserved motif and gene structures of CcPUBs

The conserved motifs were evaluated using the program MEME. Ten motifs were identified in CcPUBs, and the distribution pattern of these motifs were distinct in different 9 Groups. For instance, Motif 1 and Motif 2 were found in all genes of the 9 Groups, indicating that Motif 1 and Motif 2 were determined to be conserved U-box sequences, which could be necessary to maintain the U-box structure and support their ubiquitin linkage activity (Fig. 2a). The genes in the different group within the phylogenetic tree exhibited different conserved motifs, which indicated that different CcPUB maybe show different functions. For example, the CcPUB members in Group E have specific motif 9, these suggest that Group E’ CcPUB members have specific functions during the coffee growth and development. Furthermore, the genes in the same group within the phylogenetic tree exhibited similar conserved motifs, such as Group G (motif 1, motif 2, motif 3, motif 4, motif 7 and motif 8), Group H (motif 1, motif 2, motif 3, motif 4, motif 5, motif 6, motif 7 and motif 8), Group J (motif 1, motif 2, motif 3, motif 4, motif 5, motif 6, motif 7, motif 8 and motif 10), which indicated that they might have similar functions.

Fig. 2
figure 2

Structural and motif analysis of C. canephora L. PUB genes. (a) Conserved motifsof CcPUB proteins. Ten conserved motifs are represented by different color boxes. (b) Exon-intron structures of CcPUB genes. Green boxes mean exons, and black lines mean introns

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The exon-intron structure of the CcPUBs was examined. The results showed most of Group F (except CcPUB43), Group J and Group K showed relatively conserved gene structure with one exon and without intron (Fig. 2b). However, the gene structure in Group E (2 ~ 16 exons) varies dramatically. As showed in Fig. 2b, the intron or exon numbers and lengths in different subgroups were different. Loss or gain of the exon/intron took place during the evolution of PUB genes family in coffee, especially in Group E. Our results suggested that PUB genes maintained a relatively constant exon-intron composition during evolution of the Coffea canephora genome. In addition, analysis of gene structure and protein motifs further supported the phylogenetic relationship of PUBs in Fig. 1.

Chromosomal location and homologous gene analysis of CcPUB genes

The chromosomal distribution and collinear relationship of CcPUBs in the genome were identified. As a result, 47 CcPUB genes were mapped unevenly and nonrandomly onto 11 chromosomes, and no SbPUB gene was mapped onto chromosome 8. Chromosome 02 had the most CcPUB genes of 11, followed by chromosomes 1 and 7 with 8 genes. Each of chromosomes 0, 03, 05, and 09 contained one CcPUB genes. Five PUB genes were mapped onto chromosomes 06 and 11 (Fig. 3 and Table S3). A total of 34 homologous gene pairs were identified in the S. bicolor PUB gene family, which contained 46 homologous genes. Two homologous gene pairs were detected into chromosomes 02 (CcPUB15/CcPUB14 and CcPUB13/CcPUB16) and chromosomes 7 (CcPUB36/CcPUB34 and CcPUB37/CcPUB34), one homologous gene pair was detected into chromosomes 1 (CcPUB1/CcPUB3), chromosomes 6 (CcPUB26/CcPUB27) and chromosomes 11 (CcPUB47/CcPUB44), respectively. And three homologous gene pairs were detected between chromosomes 07 and chromosomes 11 (CcPUB33/CcPUB47, CcPUB33/CcPUB44 and CcPUB34/CcPUB43) (Fig. 3 and Table S4).

Fig. 3
figure 3

The synteny relationship of PUB genes in C. canephora L. Column in blue represent chromosomes. Lines in grey represent homologous gene pairs

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To explore the expansion pattern during the evolution, we further detected the duplication type of 47 PUB genes in C. canephora L. by MCScanX. The results showed that the WGD event accounted for 43 CcPUB genes, followed by two dispersed duplication events (CcPUB12 and CcPUB14), and one tandem duplication event (CcPUB27) and one singleton duplication event (CcPUB31) (Table S5). Therefore, the dispersed and WGD duplication events (approximately 95.7%) may have resulted in the expansions of the PUB gene family in Coffea canephora. It is thought that most duplicated genes will be silenced over time, but there are still a few maintained by purifying selection. These results showed WGD duplication events might play a crucial role in the expansion of the PUB gene family in Coffea canephora genome.

Cis‑acting elements predication of CcPUB genes

We further investigated the function of the CcPUB genes, we predicted the cis-acting elements of the putative promoter regions of the CcPUB genes using the PlantCARE database and examined the response of the cis-elements to stresses, phytohormone responsive, and plant growth and development (Fig. 4). The cis-acting elements which were the binding regions of transcription factors played a crucial role in regulating gene expression. The results showed that all of 47 PUB genes had cis-elements related to stress and plant growth and development, while 37 PUB genes had cis-elements related to phytohormone responsive. Abiotic and biotic stresses responsive cis-elements included DRE core, MBS, MYB, MYC, STRE, W box, and ABRE. Phytohormone responsive cis-elements was as-1-element cis-elements. Plant growth and development responsive cis-elements included AE-box, CAT-box, GA-motif, GATA-motif, G-box, GCN4-motif, GT1-motif, I-box, and TCT-motif. In addition, the promoter region of CcPUB6 contained most abiotic and biotic stresses responsive cis-elements (24 cis-elements), and the promoter region of CcPUB35 contained least abiotic and biotic stresses responsive cis-elements (five cis-elements). The promoter region of CcPUB22 contained most ABRE cis-elements (12 cis-elements) which was abiotic and biotic stresses responsive, and most G-box cis-elements (seven cis-elements) which was plant growth and development responsive. These results indicating that the PUB gene family may participate in a variety of biological processes.

Fig. 4
figure 4

The cis-acting elements analysis of putative promoter of 47 CcPUB genes. (a) The distribution pattern of 17 cis-acting elements of putative promoter of PUB gene family in C. canephora L. The color scale at the top right indicated the number of cis-acting elements. (b) The number of 17 cis-acting elements of putative promoter of CcPUB genes. Green color indicated the number of cis-acting elements on abiotic and biotic stresses, orange color indicated the number of cis-acting elements on phytohormone responsive, blue color indicated the number of cis-acting elements on plant growth and development. 17 cis-acting elements including: DRE core, MBS, MYB, MYC, STRE, W box, ABRE, as-1, AE-box, CAT-box, GA-motif, GATA-motif, G-box, GCN4-motif, GT1-motif, I-box, and TCT-motif

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Expression profiles analysis of CcPUB genes in tissue-specific based on RNA-Seq dataset

The expression patterns of CcPUB genes in three tissues (leaves, stems, and fruits) were analyzed using RNA-seq data. We used RPKM (reads per kilobase per million) values to estimate the expression level of CcPUB genes. The expression patterns of 47 CcPUBs in three tissues (leaves, stem and fruits) were analyzed, the results showed that the expression of CcPUBs were varied (Fig. 5). For instance, Among the 47 CcPUB genes, 40 genes (85.11%) were expressed in leaves tissue, 33 genes (70.21%) were expressed in stem tissue, only 14 genes (29.78%) were expressed in fruit tissue. We found 5 CcPUB genes (CcPUB13,CcPUB22,CcPUB33,CcPUB34, and CcPUB40) were expressed in all three diferent tissues, indicating that they have various roles in the growth development of plants. So, we referred that these five PUB genes might particular in resistance in the process of coffee growth and development. Interesting, we found 27 PUB genes exhibited highest expression (RPKM value less than 1) in leaf, suggesting that these 27 genes might involve the development of leaf. Due to leaf is an important plant organ involved resistance, we referred that these 27 PUB genes might play an important role in resistance to stresses during coffee life. Moreover, four genes (CcPUB2, CcPUB10, CcPUB24, and CcPUB45) were low expressed in all tissues. Approximately 25 non-expressed PUB genes (RPKM value less than − 1) were identifed in fruit tissues, and they may lost the function during the evolution process of PUB gene family in Coffea canephora fruit.

Expression analysis of selected CcPUB genes under stresses based on qRT-PCR

To explore the functions of PUB gene family in C. canephora L., we detected the CcPUBs expression level in leaves of seedling samples in greenhouse under four different stresses, including dehydration, low temperature, SA and Colletotrichum. We randomly selected 10 CcPUB genes were to perform qRT-PCR. Then, 10 genes are comprised of CcPUB2 (Group A), CcPUB9 (Group H), CcPUB13 (Group J), CcPUB14 (Group I), CcPUB24 (Group E), CcPUB30 (Group K), CcPUB33 (Group G), CcPUB34 (Group F), CcPUB40 (Group E), and CcPUB45 (Group D).

Under dehydration stress, two PUB genes (CcPUB2 and CcPUB40) were significantly up-regulated expressed (Fig. 6a). However, CcPUB24 and CcPUB30 was not significantly differentially expressed under dehydration stress. Among the 8 up-regulated genes, CcPUB9, CcPUB13 and CcPUB14 were up-regulated expressed to the peak levels at 1 h dehydration treatment and recovered to normal levels at 9 h. CcPUB2 and CcPUB40, exhibited highest expression level at 12 h, where CcPUB33 exhibited highest expression level at 9 h under dehydration treatment. In addition, CcPUB40 exhibited expression level more than 30 times at 12 h. These results suggested that CcPUB9, CcPUB13 and CcPUB14 respond to dehydration treatment faster than that of CcPUB2, CcPUB33 and CcPUB40. Terefore, PUB gene family in pear play vital role in the process of dehydration stress response.

Under low temperature stress (Fig. 6b), we found that seven genes (CcPUB2, CcPUB9, CcPUB13, CcPUB14, CcPUB33, CcPUB34 and CcPUB45) were up-regulated at the first half of time, and then down-regulated. Like CcPUB2 and CcPUB14 were up-regulated at 9 h under cold treatment and down-regulated later. The CcPUB9, CcPUB33, CcPUB34 and CcPUB45 exhibited to peak at 6 h. In addition, CcPUB13 exhibited highest expression level at 1 h, CcPUB30 exhibited highest expression level at 12 h, where CcPUB24 and CcPUB40 exhibited highest expression level at 24 h under low temperature treatment. And CcPUB24 exhibited expression level more 250 times at 24 h than that at 0 h.

Fig. 5
figure 5

The expression pattern analysis of CcPUB gene family in three different tissues. The heatmap of expression level of CcPUBs in three different tissues, including stem, fruit and leaf. Pheatmap, an R package, were used to generate the heatmap. The color scale represents log2(RPKM + 1). Yellow color represented high expression, while blue represented low expression

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Fig. 6
figure 6

The expression levels of 10 randomly selected CcPUBs in C. canephora L. leaves under different stresses. (a) Expression profiles of ten candidate CcPUB genes under drought treatment. (b) Expression profiles of ten candidate CcPUB genes under low temperature stress. (c) Expression profiles of ten candidate CcPUB genes under SA treatment. (d) Expression profiles of ten candidate CcPUB genes under C. fructicola stress. The values on X axis and Y axis represent the time point (hour) and the relative gene expression levels, respectively. The error strip represents three independent standard deviations (SD). Biological repetition

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In the SA treatment (Fig. 6c), five CcPUB genes (CcPUB2, CcPUB13, CcPUB24, CcPUB34 and CcPUB45) were signifcantly up-regulated expressed under SA stress treatment. CcPUB2 and CcPUB13 exhibited highest expression level at 24 h, CcPUB2 exhibited expression level more 100 times at 24 h than that at 0 h. The CcPUB34 and CcPUB45 exhibited highest expression level at 3 h, and CcPUB24 exhibited highest expression level at 6 h under salt treatment. These suggest that CcPUB34 and CcPUB45 genes respond to SA treatment actively. The CcPUB9 was down-regulated expressed during 12 h, then was up-regulated at 24 h after SA treatment.

In Colletotrichum treatment (Fig. 6d), all of 10 PUB genes were respond to the Colletotrichum fructicola stress. And two genes (CcPUB9 and CcPUB30) were down-regulated at first, then were up-regulated at 9 h after Colletotrichum treatment. The expression of CcPUB2, CcPUB13 and CcPUB40 was highest at 9 h. The expression of CcPUB9 and CcPUB14 was highest at 12 h, while the expression of CcPUB24, CcPUB30, CcPUB33, CcPUB34 and CcPUB45 was highest at 24 h. Especially, CcPUB24 exhibited expression level more 140 times at 24 h than that at 0 h. under Colletotrichum treatment.

Subcellular localization of selected CcPUB protein

Because CcPUB2, CcPUB24, CcPUB34 and CcPUB40 showed highest expression level in Coffea canephora leaves under SA, low temperature, Colletotrichum and dehydration stress, respectively. To further detect the function of CcPUB genes in coffee, CcPUB2, CcPUB24, CcPUB34 and CcPUB40 were selected to perform subcellular localization experiment. In N. benthamiana, CcPUB2-YFP and CcPUB34-YFP was located in the plasma membrane and nucleus, and CcPUB24-YFP and CcPUB40-YFP was located in the nucleus (Fig. 7). The well-characterized PUB members, including GhARM144 (contain an armadillo (ARM) repeat domain and an U-box domain) from G. hirsutum [57], and OsPUB41 from O. sativa [59], were the positive marker for subcellular localization (Fig. S1).

Fig. 7
figure 7

Subcellular localization of selected CcPUBs in N. benthamiana leaves. CcPUBs-YFP were expressed in N. benthamiana leaves, the results show that CcPUB2-YFP and CcPUB34-YFP was located in the plasma membrane and nucleus, and CcPUB24-YFP and CcPUB40-YFP was located in the nucleus. YFP was used as the control. Red scale bar = 50 μm, blue scale bar = 100 μm

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In addition, these four representative genes were selected for sequence and structure analysis. The Phyre2 database was used to predict and analyze CcPUBs’ secondary and tertiary protein structures [60]. They all possessed typical domains: RING_Ubox domain. From 633aa to 681aa of CcPUB2, 58aa to112aa of CcPUB24, 33aa to 84aa of CcPUB34 and 277aa to 303aa of CcPUB40 were the RING_Ubox domain. Furthermore, CcPUB2, CcPUB24, CcPUB34 and CcPUB40 have no signal peptides and transmembrane domains (Fig. S2a). The secondary and 3D structures indicated that CcPUBs are relatively structurally varied (Fig. S2b). RING structure consisting of three beta-sheets and a single alpha-helix, which would be stabilized by salt bridges instead of chelated metal ions. The 3D structures of CcPUB2 and CcPUB34 correspond to SAUL1 (a PUB-ARM protein SENESCENCE-ASSOCIATED E3 UBIQUITIN LIGASE1) [61], and The 3D structures of CcPUB24 and CcPUB40 correspond to UBXD1 [62]. The above results indicated that PUBs play different important roles throughout the coffee life cycle.

Discussion

Coffee, native to Ethiopia, originated from “Kaffa”. The two most widely cultivated species of coffee are C. arabica L. and C. Canephora L, (robusta). Of the total world coffee production, C. arabica L. accounts for 66% and C. canephora L. only of 34% [63]. In China, C. arabica has been widely cultivated in Yunnan province, while C. Canephora L. in Hainan province. Coffee acts as a rising cash crop, it has become the important agricultural products to help people making more income in Chinese tropical regions [64]. Coffee production industry have been threatened by many challenges in nature, including biotic and abiotic stresses. The fungal pathogens, especially Colletotrichum species, and Fusarium species are causing a major yield loss in coffee production industry [63]. Coffee leaf rust caused by Hemileia vastatrix is a very serious problem in the production of this crop [65]. Drought is a widespread limiting factor and affects flowering and bean development, hence coffee yield [63]. Although molecular mechanisms of stresses tolerance have been widely studied in model plants [66], they are less well understood in Coffee.

The U-box genes, a family of ubiquitin ligases, encode a conserved U-box motif which has 70 amino acids, they belong to the ubiquitin ligase family, and they can regulate the ubiquitination of substrates [3]. U-box genes were widely distributed in the plants. U-box genes were identified to participate in many biological processes including plant hormone signaling regulations [67], biotic stress [6, 30, 68] and abiotic stress [69, 70]. Due to the functional of PUB genes during plant development, PUB genes have been identified in many plant species. In this study, 47 PUB genes were identified in robusta using bioinformatics analysis, and the number of PUB genes in C. Canephora L. is similar to that of T. cacao (56 members), V. vinifera (49 members), A. trichopoda (37 members).

The results of CcPUB genes phylogenetic tree in this study were different from other species [20, 23]. Previous study showed that 125 S. bicolor PUB proteins were classified into six groups using phylogenetic tree analysis [19], and 69 PUB genes of M. domestica were divided into seven groups [71]. In our study, phylogenetic relationship analysis showed that CcPUBs displayed closer relations with VvPUBs. CcPUB genes were classified into nine groups based on both phylogenetic tree, motif analyses, and gene structure analyses. Interestingly, the results of motif analysis showed that CcPUBs were classified into nine groups like the phylogenetic tree, and similar structures genes were cluster into the same groups. We also found that many CcPUBs encoded some other conserved domains, such as ARM domain. For example, CcPUB9 contained five ARM domains, CcPUB13 contained one ARM domains, CcPUB14 contained seven ARM domains, and CcPUB30 contained two ARM domains. Previous studies showed that ARM repeat at the C-terminus could regulate plant development and response to stresses [57, 72]. GhARM144, which encodes a C-terminal ARM repeat domain and an N-terminal U-box domain, negative regulate the immunity against cotton Verticillium wilt [57].

Gene duplication is a mainly pathway of gene family expansion [12, 73]. Gene duplication contain whole-genome duplication (WGD), tandem, and segmental duplication. These gene duplication have played important roles in gene family evolution. The duplicated genes displayed the extended functions during the plant development and growth [28]. For example, StU-box 6 and 11 were involved in tandem duplication, and may play a key role in plant growth in S. tuberosum [26]. In our research, the WGD was the mainly gene duplication, 44 CcPUB genes were involved in WGDs. These results suggested the gene family expansion of PUB gene family in C. canephora L. mainly rely on WGD. The paralogous copies play an important factor in the occurrence of gene clusters and diversity distribution in the U-box family [21]. Based on the evolutionary history of genes, homologous gene have similar functions reflecting their conserved domains [26]. In this study, the syntenic analysis showed there were 34 homologous gene pairs in CcPUB gene family. In addition, we found that PUB genes which have similar functions and structural domains displayed a trend to cluster in the same subfamilies. Although, supplementary investigation is required to examine the particular function of one gene.

Analysis of cis-acting elements of putative promoters of the CcPUB genes, we found that CcPUB genes were commonly associated with plant growth and development, phytohormone responsive, abiotic and biotic stresses-response factors. In A. thaliana, the SA-induced PATHOGENESIS RELATED (PR)-1 promoter is regulated through the two TGACG motifs of the so called as-1 (activation sequence-1)-like element [74]. There were 37 CcPUB genes that contained salicylic acid response as-1 elements. These results suggested that the PUB gene family may take participate in regulating SA signaling pathways. In Solanum melongena, the SmCP promoter with a G-box increased S. melongena cysteine proteinase expression in senescent fruits and circadian-regulated leaves [75]. In O. sativa variety Minghui 63, G-box was identified cis-elements as positive regulators of senescence [76]. In our study, 22 CcPUBs promoter have G-box, CcPUB22 has 7 G-box. CcPUBs may play an important in plant growth and development, especially CcPUB22. GT1-motif and I-box were the light-responsive promoter motifs [77, 78]. The 32 of 47 CcPUBs promoter have GT1-motif, and CcPUB37 promoter have both GT1-motif and I-box. The W box cis-elements in the promoters of their target genes have been implicated in biotic and abiotic stress responses, such as those triggered by pathogens [79], drought [80], cold [81], and heat stress [82]. The MYC motif within the promoter region of GhTIP1;1-like were the core cis-elements in response to low temperature in G. hirsutum [83]. GhMYB102 could directly bind the MYB motif elements in the promoter regions of GhNCED1 and GhZAT10 to positive regulate the drought tolerance of G. hirsutum [84]. In our research, all of CcPUBs promoter regions have MYC motif, 46 of 47 CcPUBs promoter regions have MYB motif, and 30 of 47 CcPUBs promoter regions have W box. In addition, one CcPUB promoter regions contains many different motifs. These elements were shown to rapidly regulate gene expression in response to changes in nature. These findings help us better understanding the molecular mechanisms of CcPUB gene functions.

To verify these CcPUB genes, 10 genes were selected to perform qRT-PCR under various stresses, including dehydration, low temperature, SA and Colletotrichum stress. Previous studies have reported that PUB genes involved in stress responses [31, 32]. A large number of PUB genes were induced expression during abiotic and biotic stresses [24, 25, 27, 85]. In this study, CcPUB40 was significantly up-regulated under dehydration treatment, CcPUB24 was significantly up-regulated expressed under low temperature treatment, CcPUB2 was significantly up-regulated under SA treatment, and CcPUB34 was significantly up-regulated under Colletotrichum treatment. Based on RNA-seq data, CcPUB2, CcPUB24, and CcPUB40 showed low expression level in coffee leaves. These results suggested these four genes CcPUB40, CcPUB24, CcPUB2, and CcPUB34 could response to dehydration, low temperature, SA and Colletotrichum stress, respectively. In addition, the results of subcellular localization experiment of CcPUB2, CcPUB24, CcPUB34, and CcPUB40 indicated that CcPUB genes might act biology function at different cell localization. U-box proteins are widely distributed among eukaryotic organisms and show a higher prevalence in plants than in other organisms. RING finger/U-box-containing proteins are a group of diverse proteins with a variety of cellular functions, including oncogenesis, development, viral replication, signal transduction, the cell cycle and apoptosis [3, 8]. But the mechanism of CcPUB genes, such as CcPUB2, CcPUB24, CcPUB34, and CcPUB40 regulating stressses responses remained unclear, and needed to be explored in the future study.

Conclusion

In our study, a total of 47 CcPUB genes were identified in Coffea canephora (Robusta) genome. These 47 CcPUBs were divided into nine groups in phylogenetic tree. The results of conserved motifs and gene structures analysis were consistent with the result of phylogenetic tree. Cis-acting element analysis indicated that PUB genes might participate in plant growth and development, phytohormone responsive, abiotic and biotic stresses-response. RNA-seq data from three tissues exhibited different expression level of CcPUB genes. The qRT-PCR analysis was used to identify selected genes associated with abiotic and biotic stresses. In addition, CcPUB2, CcPUB24, CcPUB34, and CcPUB40 were selected to further study. Subcellular localization showed CcPUB2 and CcPUB34 were located in the plasma membrane and nucleus, and CcPUB24 and CcPUB40 were located in the nucleus. But the cellular mechanism of CcPUB2, CcPUB24, CcPUB34, and CcPUB40 regulating stresses responses was needed to be explored in the future study.

Data availability

Data is available at NCBI SRA, accession numbers: PRJNA561881 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA561881) and PRJNA798825 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA798825).

Abbreviations

PUB:

Plant U-box

SA:

Salicylic acid

RNA:

Ribonucleic Acid

DNA:

Deoxyribo Nucleic Acid

qRT-PCR:

Quantitative Real-Time Polymerase Chain Reaction

ATP:

Adenosine triphosphate

YFP:

Yellow fluorescent protein

ARM:

Armadillo

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Acknowledgements

We thank Dr. Lisen Liu (Institute of Cotton Research, Chinese Academy of Agricultural Sciences) for kindly providing us with pCAMBIA2300-YFP vector for subcellular localization, Dr. Fei Wei (Institute of Cotton Research, Chinese Academy of Agricultural Sciences) for kindly providing us with the GhARM144-GFP for well-characterized PUB members subcellular localization, Dr. Cheng Li (School of Life Sciences, Henan University) for kindly providing us with the OsPUB41-GFP, p2300-35 S-H2B-mCherry-OCS and pBI221-mCherry-PM for well-characterized PUB members subcellular localization.

Funding

This work was supported by the Hainan Provincial Natural Science Foundation of China (No.322QN401), the Central Public-interest Scientific Institution Basal Research Fund (No.1630142024002) and the Special Fund for Young Talents in Henan Agricultural University (No. 30501338).

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Author notes

  1. Shichao Liu, Ruibing Liu and Pengyun Chen contributed equally to this work.

Authors and Affiliations

  1. Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences, Wanning, Hainan, 571533, China

    Shichao Liu, Ruibing Liu, Shengfeng Gao, Yafeng Gou, Tian Tian, Siwei Wen & Shiwei Sun

  2. College of Agronomy and Biotechnology, China Agricultural University, Beijing, 100193, China

    Pengyun Chen

  3. College of Plant Protection, Henan Agricultural University, Zhengzhou, 450002, China

    Bo Chu & Chenchen Zhao

  4. Key Laboratory of Genetic Improvement and Quality Regulation for Tropical Spice and Beverage Crops of Hainan Province, Wanning, Hainan, 571533, China

    Shichao Liu, Shengfeng Gao, Yafeng Gou & Shiwei Sun

  5. Key Laboratory of Genetic Resource Utilization of Spice and Beverage Crops, Ministry of Agriculture and Rural Affairs, Wanning, Hainan, 571533, China

    Lin Yan

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  1. Shichao Liu
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Contributions

SL, RL and PC: Conceptualization, Methodology, Software, Writing. BC and SG: Methodology, Software. YG and TT: Software, Visualization, Data curation. SW and LY: Visualization, Investigation. CZ and SS: Review & editing. SL, TT and CZ: Funding acquisition.

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Correspondence to Chenchen Zhao or Shiwei Sun.

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Liu, S., Liu, R., Chen, P. et al. Genome-wide identification and expression analysis of the U-box gene family related to biotic and abiotic stresses in Coffea canephora L.. BMC Genomics 25, 916 (2024). https://doi.org/10.1186/s12864-024-10745-w

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Keywords

BMC Genomics

ISSN: 1471-2164

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