1. Introduction
Plants consistently encounter diverse adverse ecological stress factors, including extreme temperatures, drought, and saline-alkali stress during growth and development [1]. Calcium ion (Ca2+), functioning as second messengers, can detect and transmit external stress signals within plants, as well as participate in various signal transduction processes [2]. When plants perceive external stimuli, the Ca2+ concentration in the cytoplasm rapidly increases. The free Ca2+ binds to calcineurin B-like protein (CBL), a Ca2+ sensor, to regulate downstream target proteins, triggering physiological changes in plants and contributing to the response to external environmental stimuli [3]. To date, four types of Ca2+ receptors have been identified, including calmodulin protein (CaM), calmodulin-like proteins (CML), calcium-dependent protein kinase (CDPK), and CBL [4]. Previous research has demonstrated that CBL members possess a distinct ability to bind Ca2+, which can bind to the serine/threonine of CIPK and activate target proteins. This activation, in turn, catalyzes the phosphorylation of various target proteins to generate feedback in response to stress [5]. This pathway regulates multiple processes, including defense responses, ion homeostasis, and gene expression [6].
Previous studies have established that CBL proteins contain four elongation factor hand (EF-hand) structures, which are conserved domains capable of binding Ca2+ [6]. Analysis of CBL amino acid sequences in Arabidopsis thaliana, revealed variability in the number of typical EF-hand domains. Specifically, AtCBL6, AtCBL7, AtCBL8, and AtCBL10 contain only one typical EF-hand domain, while AtCBL1 and AtCBL9 possess two. Notably, AtCBL2, AtCBL3, AtCBL4, and AtCBL5 lack typical EF-hand domains [2]. The diversity in domains among CBL proteins may result in district Ca2+-binding capabilities, providing a foundation for CBLs’ response to various stimulus signals that affect Ca2+ concentration [7]. Furthermore, CBL proteins also contain an FPSF motif. The spatial structure of this motif enables specific recognition and binding to the corresponding structural regions on interacting protein kinases [8].
The first CBL gene was identified in Arabidopsis thaliana [9]. To date, the CBL gene family has been characterized across diverse species., including Oryza sativa [10], Solanum lycopersicum [11], Gossypium hirsutum Linn [12], Vitisvinifera [13], and Ananas comosus [14]. CBLs play crucial roles in plant growth and development processes, such as seed germination and pollen tube germination, as well as in response to abiotic stresses, including high salinity, low temperature, and drought [15,16]. The expression patterns of CBL transgenic plants vary under different abiotic stresses, thereby modulating plant stress tolerance [15]. Previous studies have demonstrated that CBL genes enhance stress tolerance in various plants. The roles of CBLs in regulating abiotic stresses, particularly drought and salt stress, have been well-documented. Overexpression of CBL1, CBL9, CIPK1, and CIPK6 has been shown to increase drought tolerance in Arabidopsis [17]. Heterologous expression of AcCBL1 (Ananas comosus CBL11) and AcCIPK5 enhances tolerance to salt, osmotic, and cold stresses in transgenic Arabidopsis [14,18]. GmCBL1-OE lines improve alkaline tolerance in transgenic soybeans by regulating redox reactions [19]. Conversely, atcbl10 mutation leads to increasing salt sensitivity in Arabidopsis [20]. AtCBL2 and AtCBL3 participate in growth, embryo, and seed development, as well as response to high-concentration ion stress in Arabidopsis [7]. AtCBL2 responds to high alkaline (high pH value) stress by binding to AtCIPK11 [21]. AtCBL4 promotes the antiporter AtSOS3 to pump excessive Na+ out of cells, ensuring intracellular Na+ balance by interacting with AtCIPK24 [22].
The distribution area of saline-alkali land in Northeast China is approximately 765 hm2 [23]. P. simonii × P. nigra, a hybrid poplar characterized by its fast growth and strong adaptability to the external environment, has been widely used for afforestation and commercial forestry in Northeast China. The genome-sequenced tree species provide a research foundation for the functional study of the CBL gene in P. simonii × P. nigra. In this study, PtrCBLs were identified and analyzed from P. trichocarpa through bioinformatics methods, including analyses of protein structures, evolutionary relationships, chromosome localization, protein interaction networks, and protein phosphorylation site prediction. Transcriptome technology, serving as a powerful bioinformatics tool, plays a crucial role in contemporary biological research. We employed RNA-seq and RT-qPCR techniques to analyze the expression patterns of PsnCBLs in P. simonii × P. nigra under different alkaline concentration treatments. Our predictions and experimental results will provide a preliminary basis for further functional research on CBL genes under alkaline stress in P. simonii × P. nigra and other tree species.
2. Materials and Methods
2.1. Identification of CBL Genes in P. trichocarpa
To identify the CBL members in P. trichocarpa, we obtained 10 AtCBL genes from the Arabidopsis Information Resource (TAIR10) website (
2.2. Phylogenetic Analysis and Multiple Sequence Alignment
We obtained 15 GmCBL IDs, as referenced in a previous study, and acquired amino acid sequences from the Phytozome database [19]. The amino acid sequences of 13 PtrCBLs, 10 AtCBLs, and 15 GmCBLs were aligned using ClustalW in MEGA11 (v11.1.10) [30]. The Neighbor-Joining method was employed to construct the phylogenetic tree with 1000 bootstrap replications. We utilized the Gene Structure Display Server website to visualize the distribution of introns and exons using genomic sequences and coding sequences [31]. The Multiple EM for Motif Elicitation (MEME, v5.5.7,
2.3. Analysis of Promoter cis-Acting Elements
The promoter regions of PtrCBLs were defined as 2000 bp upstream of the coding sequence, including the 5′UTR. The PlantCARE online tool (
2.4. Prediction of Phosphorylation Sites
The amino acid sequences of PtrCBLs were used as the input for the prediction of phosphorylation sites using the PtrNetPhos 3.1 website (
2.5. Protein Tertiary Structure and Interaction Network
The protein tertiary structure of the PtrCBLs was analyzed using the SWISS-MODEL online platform (
2.6. Plant Materials, Alkaline Stress and RNA-Seq Analysis
The branches of two-year-old P. simonii × P. nigra, cultivated at Jilin Agricultural Science and Technology University, Jilin, China, were cut into 10 cm cuttings and inserted into flowerpots containing a substrate mixture (humus soil:vermiculite:perlite = 5:3:2) for cultivation in April 2024. The cultivation was conducted at 24 ± 2 °C, with a 16-h light period (5000 Lux) and an 8-h dark period in a greenhouse. Plants were irrigated with Hoagland nutrient solution, applying 250 mL to each flowerpot twice weekly. After 4 weeks of culture, P. simonii × P. nigra exhibiting consistent growth were selected for alkali stress treatment. The alkali stress solution comprised Na2CO3 and NaHCO3 in a 1:1 ratio. Hoagland nutrient solutions containing three different alkali concentrations, including 0 mmol/L, 50 mmol/L (low concentration), and 200 mmol/L (high concentration), were prepared to irrigate the P. simonii × P. nigra. The group with 0 mmol/L (Hoagland nutrient solution without Na2CO3 and NaHCO3) served as the control. Each flowerpot received 250 mL of solution, with the control group receiving an equal volume of water concurrently. Irrigation occurred twice weekly, with three biological replicates per treatment group. Leaves of P. simonii × P. nigra were sampled at 7 d and 14 d after each treatment, respectively, rapidly frozen in liquid nitrogen, and stored at −80 °C for subsequent analysis.
RNA sequencing was conducted by BioMarker Technologies Company (Beijing, China). The experimental procedure involved constructing mRNA libraries for each P. simonii × P. nigra sample under alkaline stress, followed by sequencing using the Illumina Novaseq platform. Upon completion, the raw data underwent quality assessment. This process involved removing reads containing adapter sequences, poly-N fragments, and those of poor quality, ensuring high-quality data for subsequent analyses and guaranteeing accurate and reliable results. HISAT2 software (version 2.2.1) was employed to align the high-quality reads with the P. trichocarpa reference genome, ensuring accurate read localization on the genome. The feature Counts software (version 1.5.0) was used to quantify the aligned reads corresponding to each gene, with the FPKM value serving as the standard quantitative unit for gene expression levels. Differentially expressed genes (DEGs) were identified using the DESeq2 analysis tool. The screening criteria for DESeq2, differentially expressed genes (DEGs) were as follows: genes with a false discovery rate (FDR) below 0.01 and an absolute value of log2 (fold change) not less than 1.2 [35]. The processed data were visualized as expression heatmaps using GraphPad Prism 10, with expression levels calculated by log2 (FPKM).
2.7. RNA Extraction and RT-qPCR
Fresh leaves from each treatment were harvested, frozen in liquid nitrogen, and stored at −80 °C until RNA isolation. High-quality RNA was extracted from P. simonii × P. nigra using the RNeasy Plant Mini Kit (Qiagen, Dusseldorf, Germany), following the manufacturer’s instructions. RNA quality was assessed using 1.2% agarose gel electrophoresis and a nucleic acid concentration analyzer (Nanodrop 2000, Thermo Scientific, Waltham, MA, USA). The results of the RNA quality check are provided in Supplementary Table S2 and Supplementary Figure S1. RNA was reverse transcribed to cDNA using the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen, Beijing, China). RT-qPCR was performed using a 10 μL reaction system with AceQ® Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China). The RT-qPCR conditions were as follows: initial denaturation at 95 °C for 7 min, followed by 35 cycles of 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 30 s. The qTOWER 3G Cycler (Analytik Jena, Jena, Germany) was used for the RT-qPCR reaction, and the relative expression patterns of the PsnCBL genes were calculated using the 2−∆∆Ct method. Nine PsnCBL (PsnCBL2, 3, 4, 5, 6, 8, 10, 12, and 13) with relatively high FPKM values in the transcriptome were selected for primer design. Primers were designed within the 5’UTR using the NCBI Primer-Blast online tool. The PsnActin gene (GenBank ID: XM_002298946) served as the internal reference gene [36]. All primer information for PsnCBL and PsnActin is provided in Supplementary Table S3.
2.8. Statistical Analysis
A two-tailed Student’s t-test method in SPSS 22.0 software was used to verify the results of RT-qPCR. An asterisk (*) represents a significant difference (p < 0.05), and double asterisks (**) represent an extremely significant difference (p < 0.01). All data were obtained from at least three biological replicates. Subsequently, the data are presented in the form of means ± standard deviation (SD).
3. Results
3.1. Identification of the CBL Genes in Populus trichocarpa
In this study, 13 PtrCBL family genes in P. trichocarpa were identified and named PtrCBL1-PtrCBL13. Physicochemical property analysis of the PtrCBLs revealed amino acid sequences ranging from 213 aa to 254 aa. PtrCBL8 exhibited the largest molecular weight at 29.24 kDa, followed by PtrCBL4 at 27.83 kDa, while the remaining 11 PtrCBL genes ranged from 24.01 kDa to 25.89 kDa. Theoretical pI values ranged from 4.59 to 4.94, indicating that all PtrCBLs were acidic proteins. The aliphatic index varied from 85.96 to 106.76. The GRAVY of PtrCBLs ranged from −0.007 to −0.305, suggesting their hydrophilic nature. More than half of the PtrCBL genes (PtrCBL3, 4, 7, 8, 9, 10, 11, and 13) demonstrated an instability index greater than 40, indicating potential instability and susceptibility to changes in physical, chemical properties, and biological functions. Additionally, PtrCBL4 and PtrCBL8 each contained one transmembrane domain. Predicting the subcellular localization of the PtrCBL proteins contributes to understanding their molecular functions. The analysis revealed that almost all PtrCBL genes were localized to the cell membrane, except for PtrCBL6, which was exclusively found in the cytoplasm. This unique localization suggests that PtrCBL6 may perform specialized protein functions. Detailed information on PtrCBLs is presented in Table 1.
3.2. Chromosome Localization and KaKs Calculation
Based on the genome annotation information of P. trichocarpa, the 13 PtrCBL genes were distributed across seven distinct chromosomes. Three PtrCBL genes were located on each of Chromosome 1 and Chromosome 3. Two PtrCBLs were positioned on each of Chromosome 6 and Chromosome 12. PtrCBL9 was situated on Chromosome 11, PtrCBL10 on Chromosome 12, and PtrCBL13 on Chromosome 16 (Figure 1).
To analyze the selection pressures endured by the PtrCBL genes, we calculated the nonsynonymous substitution rate (Ka), the synonymous substitution rate (Ks), and the Ka/Ks ratio for five PtrCBL gene pairs. We used the formula T = T = Ks/2 × 6.1 × 10−9 Mya to calculate the time (T) of the duplication events [37]. The results showed that, except for PtrCBL4-PtrCBL8, for which no values could be calculated, the Ka/Ks ratios of the other four gene pairs ranged from 0.0751 to 0.2050, indicating that the PtrCBL genes have undergone purifying selection. The formation times of these segmental duplication gene pairs were approximately 4.97 to 6.81 Mya (Table 2). Taken together, these results suggest that the PtrCBL genes have exhibited a conservative evolutionary trend during the evolutionary process.
3.3. Phylogenetic Analysis
The analysis of homologous genes provides a foundation for exploring gene functions. The phylogenetic analysis revealed that the PtrCBL genes could be categorized into five subfamilies (Group I–V). Each subgroup exhibited uneven distribution in terms of member count. Group I contained the largest number of PtrCBL members (PtrCBL1, 6, 10, 11, and 12). PtrCBL4 and 8 were located in Group II, while PtrCBL2 and 5 were found in Group III. Group IV comprised two PtrCBL genes, including PtrCBL3 and 9. Both PtrCBL7 and 13 were grouped in Group V (Figure 2). It is well established that proteins with closer genetic relationships often exhibit similar or closely related biological functions. Furthermore, the majority of PtrCBL proteins clustered with the CBL proteins in Glycine max. This clustering pattern suggests that the PtrCBL proteins have a closer genetic relationship with their homologous counterparts in Glycine max compared to those in Arabidopsis thaliana.
3.4. Gene Structure and Conserved Motifs
Analysis of gene structure can elucidate conserved characteristics and potential biological functions. Multiple sequence alignment revealed that all PtrCBL proteins contained EF-hand domains 1–4. Twelve PtrCBL proteins exhibited the FPSF motif at the C-terminus, with the exception of PtrCBL6. Notably, the N-terminal MGCXXSK/T motif, recognized as the myristoylation domain, is a specialized CBL protein domain [38]. This domain was present only in PtrCBL2, 5, 10, 11, and 12 (Figure 3).
Analysis of gene structure revealed similarities in length, exon count, and intron number among members within the same subfamily (Figure 4A). Notably, all PtrCBL members, except PtrCBL8, contained seven introns, indicating conservation in the evolution of PtrCBLs (Figure 4B). Furthermore, the number of introns often correlates with gene expression level, suggesting these genes may be highly expressed in cells [39]. Conserved motifs shared by most members of a gene family typically play crucial roles in the family’s function or structure. Analysis of conserved motifs demonstrated that all PtrCBLs contained motifs 1, 2, 3, and 5, which are characteristic of conserved EF-hand domain structures. Motif 10 was unique to PtrCBL4 and PtrCBL8, while PtrCBL6 lacked motif 7. Most PtrCBL proteins possessed seven motifs, although the specific types varied. PtrCBL7 and PtrCBL13 exhibited nine conserved motifs (Figure 4C). Detailed sequences of ten motifs are presented in Supplementary Table S4.
3.5. Promoter cis-Element Analysis
Promoter cis-acting elements, crucial binding regions for transcription initiation factors, play a significant role in regulating gene expression [40]. To further analyze the potential biological functions of the PtrCBLs, we examined their promoter cis-acting elements using the PlantCARE website. We identified 10 distinct types of promoter cis-acting elements in the PtrCBLs promoter region, including abscisic acid-responsive elements (ABRE), methyl jasmonate (MeJA)-responsive elements (CGTCA/TGACG), salicylic acid-responsive elements (TCA), auxin-responsive elements (TGA), drought-inducible elements (MBS), gibberellin-responsive elements (GARE/TATC-box/P-box), and low-temperature-responsive elements (LTR). Detailed cis-acting element information is provided in Supplementary Table S5. The distribution of these cis-acting elements varied considerably among PtrCBL promoters. For example, PtrCBL6 contained the largest variety of cis-acting elements, while PtrCBL11 contained only one LTR. With the exception of PtrCBL7, 9, and 11, all PtrCBLs contained varying numbers of ABRE elements, with PtrCBL2 having the highest number. Additionally, only PtrCBL3 and PtrCBL4 contained one TGA element and TATC-box, respectively (Figure 5).
3.6. Protein Tertiary Structure
The tertiary structure of PtrCBL proteins was analyzed using Swiss-Model online platform. The results revealed diverse protein tertiary structures among the 13 PtrCBL members. Notable differences in PtrCBL tertiary structures were observed, likely related to structural elements, including α-helices, β-sheets, and random coils. The similarities and differences in tertiary structures may explain the functional similarities or differences exhibited among proteins. Importantly, homologous gene pairs such as PtrCBL2-PtrCBL5, PtrCBL3-PtrCBL9, PtrCBL4-PtrCBL8, PtrCBL7-PtrCBL13, and PtrCBL10-PtrCBL11 displayed highly similar protein tertiary structures, suggesting functional redundancy among these pairs (Figure 6).
3.7. Protein Interaction Network
We utilized the STRING website to predict potential interacting proteins for PtrCBLs. The analysis revealed that all PtrCBL proteins possessed interacting protein networks, suggesting that PtrCBL members can interact with other proteins in response to plant hormone treatments and environmental stresses, as well as participate in growth and development processes in P. trichocarpa. Furthermore, PtrCBL1, 3, 4, 6, 8, 9, 10, and 13 exhibited similar interacting proteins, indicating the presence of conserved interaction domains within the PtrCBL protein (Figure 7). Previous research has demonstrated that CBL proteins can specifically bind to CBL-interacting protein kinases (CIPK) to form CBL-CIPK protein complexes. These complexes participate in the Ca2+ signal transduction pathway, enabling plants to respond to various internal and external signals [41]. Our results indicated that all PtrCBL proteins interact with different CIPK proteins, suggesting that this interaction exists in P. trichocarpa to facilitate growth, development, and abiotic stress resistance.
3.8. Prediction in Phosphorylation Sites
Prior research has demonstrated that CBL proteins can undergo phosphorylation by CIPK proteins [42]. Consequently, we predicted potential phosphorylation sites in PtrCBL proteins, focusing primarily on serine, threonine, and tyrosine sites. As illustrated in Figure 8, all PtrCBL proteins contained serine phosphorylation sites, albeit in varying numbers. For instance, PtrCBL2 and PtrCBL12 exhibited fifteen serine sites each, followed by PtrCBL3, 5, and 9, which possessed fourteen serine sites. PtrCBL8 displayed the fewest serine sites, with only eight. PtrCBL10 and PtrCBL12 contained the highest number of threonine sites. The PtrCBL protein with the least serine sites was PtrCBL2, containing only three. With the exception of PtrCBL2 and PtrCBL5, all PtrCBL proteins contained tyrosine sites, though most PtrCBL proteins only exhibited one to three tyrosine sites.
3.9. RNA-Seq and Expression Heatmap
Previous research has documented that halophytes typically exhibit specific morphological and anatomical adaptations in response to salt stress. These adaptations primarily include alterations in stomatal size, development of a multi-layered epidermis, thickening of the cuticle, early lignification, inhibited differentiation, and changes in the diameter and number of xylem vessels [43]. In this study, leaves from P. simonii × P. nigra subjected to low and high concentrations of alkaline stress were used as plant materials, with P. trichocarpa serving as the reference genome for transcriptome sequencing analysis. The analysis of transcriptome data revealed a total of 10,657 differentially expressed genes (DEGs), comprising 6613 down-regulated and 4044 up-regulated genes (Figure 9A).
To investigate the functions of PsnCBLs under low- and high-concentration alkaline stress in poplar, we analyzed gene expression levels using transcriptome data. Notably, PtrCBL1 showed negligible count values. A heatmap of expression patterns was constructed based on log2(FPKM) values. As depicted in Figure 9B, the majority of PsnCBL genes exhibited significant up-regulation or down-regulation at 7 or 14 days under low- or high-concentration alkaline stress, suggesting PsnCBLs’ involvement in the alkaline stress response in P. simonii × P. nigra. Specifically, PsnCBL2 was significantly up-regulated under low-concentration alkaline stress conditions at 7 d, while PsnCBL4, 5, and 6 were significantly down-regulated. However, after 14 d of low-concentration alkaline stress, PsnCBL3, 4, 6, 7, 8, 10, and 13 were significantly up-regulated, and PsnCBL5 was significantly down-regulated, with other genes showing no significant change. Under high-concentration alkaline stress at 7 d, the expression levels of PsnCBL4, 8, and 13 were significantly up-regulated, while four genes (PsnCBL2, 5, 9, and 10) were significantly down-regulated, and four genes showed no significant difference in expression levels. At 14 days of high-concentration alkaline stress, PsnCBL2, 3, 5, 8, and 12 were significantly up-regulated, two genes were down-regulated, and eight genes showed no significant change. Notably, PsnCBL3 and PsnCBL8 were significantly up-regulated at all three time points under both low- and high-concentration alkaline stress, indicating their important roles in the response to alkaline stress in poplar. The transcriptome data for PsnCBLs is provided in Supplementary Table S6.
Following our transcriptome analysis, we performed Gene Ontology (GO) enrichment based on the identified DEGs. The results revealed that multiple biological processes are involved in the response to alkaline stress in P. simonii × P. nigra. These processes include carbohydrate metabolic process, protein phosphorylation, iron ion transport, iron ion homeostasis, and transmembrane transport (Figure 9C–F). Notably, for high alkaline stress at 7 d and both low and high alkaline stresses at 14 d, the DEGs were predominantly enriched in the biological process of protein phosphorylation. This finding suggests that PsnCBL proteins may contribute to alkaline stress through the phosphorylation pathway.
3.10. Expression Pattern of Populus simonii × Populus nigra Under Alkaline Stress
To validate the transcriptome data reliability, RT-qPCR was employed to access the expression of nine PsnCBLs (PsnCBL2, 3, 4, 5, 6, 8, 10, 12, and 13) with relatively high FPKM values in the transcriptome data. The evaluation revealed that the expression trends of these genes were generally consistent with the transcript abundance changes in the transcriptome data, confirming its reliability (Figure 10a,b). Further analysis under low-concentration alkaline stress showed significant up-regulation of PsnCBL3 and PsnCBL15 at 7 d and 14 d. PsnCBL4 and PsnCBL6 were markedly down-regulated at 7 d and dramatically up-regulated at 14 d. PsnCBL2 exhibited significant up-regulation at 7 d and slight up-regulation at 14 d. PsnCBL5 was significantly down-regulated at 7 d and slightly up-regulated at 14 d. PsnCBL8 and PsnCBL10 showed significant up-regulation at 14 d, while PsnCBL12 was slightly up-regulated at both 7 d and 14 d. Under high-concentration alkaline stress, only PsnCBL8 was consistently up-regulated at 7 d and 14 d. PsnCBL2 and PsnCBL5 were significantly down-regulated at 7 d and remarkably up-regulated at 14 d. PsnCBL4 and PsnCBL13 were significantly up-regulated at 7 d, with PsnCBL4 slightly down-regulated and PsnCBL13 slightly up-regulated at 14 d. PsnCBL3 and PsnCBL12 showed significant up-regulation at 14 d, while PsnCBL10 was slightly down-regulated at both 7 d and 14 d (Figure 10a,b).
4. Discussion
4.1. The CBL Family Genes in Populus trichocarpa
Calcineurin B-like proteins (CBLs), which function as plant-specific calcium sensors, are most similar to calcineurin B and neuronal calcium sensor proteins. They have been recognized as crucial components in diverse Ca2+-dependent processes in plants [44]. CBLs play a significant role in influencing biological and abiotic stress processes through Ca2+-related pathways [45]. The functions of CBL genes have been identified in various plants, including Arabidopsis thaliana [2], Oryza sativa [10], Vitis vinifera [13], and Saccharum officinarum [46]. However, until now, there has been limited bioinformatics analysis information on CBL genes in poplar. In this study, 13 PtrCBL genes were identified in the P. trichocarpa genome, all containing four EF-hand motifs. Multiple sequence alignment analysis, as shown in Figure 3, revealed that all PtrCBLs except PtrCBL6 contain the FPSF motif at the C-terminus, indicating that PtrCBL6 may be phosphorylated by CIPK through an alternative phosphorylation site, warranting further experimental verification. Previous studies have reported that the serine site in the FPSF motif can be phosphorylated by CIPK [47]. For instance, AtCIPK23 can phosphorylate the C-terminal FPSF motif within AtCBL1 and AtCBL9, thereby enhancing the interaction between CBL and CIPK proteins [8]. It is noteworthy that the 226th Ser of PtrCBL4 and PtrCBL8 was replaced with Asn in the FPSF motif. The same results were also found in Arabidopsis, in which the Ser site changed to Thr [2]. This diverges from the absolute conservation of the Ser site within the PFPF motif of OsCBLs and VvCBLs [10,13]. Our subcellular localization prediction results showed that 12 PtrCBL proteins were located on the cell membrane, while only PtrCBL6 was positioned in the cytoplasm (Table 1). However, PtrCBL1-PtrCBL6, which serve as homologous genes, were located in the cytoplasm, indicating that homologous genes or conserved domains do not necessarily share the same subcellular localization. This also suggests that homologous genes may exhibit differences in gene functions and signal transduction.
Multiple studies have demonstrated that the domains within genes play a crucial role in determining gene functions [48]. All PtrCBL proteins contain four EF-hand domains, with highly conserved connection intervals between each domain. Specifically, there are 22 amino acids between EF1 and EF2, 25 amino acids between EF2 and EF3, and 32 amino acids between EF3 and EF4 (Figure 3). These findings align closely with the CBL protein structures observed in Arabidopsis thaliana and Saccharum officinarum [2,13]. Four PtrCBL proteins (PtrCBL2, 5, 11, and 12) possess myristoylation sites (MGXXXS/T) at the N-terminus (Figure 3). N-terminal myristoylation facilitates protein attachment to membranes and enhances protein–protein interactions [49]. Furthermore, previous research has shown that myristoylation sites contribute to improved salt stress resistance [50], suggesting that PtrCBL2, 5, 11, and 12 may play significant roles in the poplar’s response to salt stress.
4.2. Promoter cis-Acting Elements of PtrCBLs
Previous research indicates that promoter cis-elements play critical roles in transcriptional regulation when plants are subjected to biotic and abiotic stresses. These elements include ABRE, MeJA-responsive elements (CGTCA), gibberellin-responsive elements (GARE-motif), auxin-responsive elements (TGA-element), P-box, LTR, salicylic acid-responsive elements (TCA-element), TC-rich repeats, TGACG motif, and drought-inducible elements (MBS cis-element). Analysis of PtrCBL promoter cis-acting elements reveals that each PtrCBL contains at least one abiotic-related stress or hormone-related responsive cis-acting element, suggesting that PtrCBLs play significant roles in responding to abiotic stress and hormone signals in poplar. All PtrCBLs, except PtrCBL9 and PtrCBL12, contain ABRE (Figure 5). The response mechanisms of CBL genes to ABA-mediated abiotic stress have been studied in Arabidopsis thaliana [51]. This phenomenon suggests that PtrCBL genes participate in the ABA-mediated signaling pathway to respond to abiotic stress.
4.3. The Predicted Protein Interaction Network of PtrCBLs
In plants, CBL proteins interact with CIPK proteins and play a crucial role in the stress response mechanism to external stimuli [52]. Protein interaction networks are significant for interpreting various Ca2+ signals to adapt to changing environments [53]. The predicted protein interaction networks demonstrated that all PtrCBL proteins interacted with CIPK proteins (as shown in Figure 6). PtrCBL2 and PtrCBL5 were homologous genes (as shown in Figure 4A). Our results indicated that both PtrCBL2 and PtrCBL5 interacted with PtrCIPK5, 7, 16, and 26, suggesting functional redundancy of homologous genes. Previous studies have shown that in the reactive oxygen species (ROS) pathway, AtCBL1 and AtCBL9 interact with AtCIPK26 and play a regulatory role in the NADPH oxidase RbohF in Arabidopsis [54]. PtrCBL2 and PtrCBL5 serve as homologous genes of AtCBL1 and AtCBL9 in Arabidopsis. Additionally, PtrCBL2 and PtrCBL5, which contain myristoylation sites at the C-terminus, can promote protein attachment to membranes and protein–protein interactions [49]. Increasing evidence has also shown that the homologous genes of PtrCBL2 and PtrCBL5 in Populus euphratica, PeCBL1, interact with PeCIPK24, PeCIPK25, and PeCIPK26 and regulate the homeostasis of Na+ and K+ in Populus euphratica [55]. This indicates that the interaction between PtrCBL2 and PtrCBL5 with CIPK proteins is of great significance in the NADPH oxidase pathway and in regulating Na+ and K+ homeostasis in poplar.
4.4. Expression Patterns and RNA-Seq Analysis of PsbCBLs Under Alkaline Stress
CBL proteins play a crucial role in metabolic and growth-related activities during plant life cycles [15]. While most studies on CBL gene functions have primarily focused on salt stress, drought, and hormone responses [56,57,58], there has been limited research on the expression patterns of CBLs under alkaline stress in poplar. Salinity and alkalinity are major abiotic stresses that impede growth and development in poplar [23]. Previous research has indicated that alkaline salts have a significantly stronger detrimental effect on plants compared to neutral salts. In saline-alkali soil containing bicarbonate ions (HCO3−), plants are subjected to the dual damaging effects of both salt and alkaline stress [59]. Consequently, soda alkaline was selected for alkaline stress treatment in P. simonii × P. nigra. Transcriptome data analysis revealed that the number of DEGs under high-concentration alkaline stress was generally greater than that under low-concentration alkaline stress, indicating that the high-concentration alkaline stress treatment caused severe damage to P. simonii × P. nigra, which aligns with previous findings [23]. The combined analysis of RT-qPCR and transcriptome data demonstrated that nine PsnCBL genes were induced under different concentrations of alkaline stress at various time points (Figure 10a,b), indicating that CBL genes play a significant role in response to alkaline stress in poplar. GO analysis showed that protein phosphorylation had the largest number of DEGs in biological processes under both low- and high-concentration alkaline stress (Figure 9C–F). This phenomenon indicates that CBL-mediated phosphorylation may trigger responses in multiple biological processes to cope with external alkaline stress in poplar. Previous research has shown that when plants are exposed to high Na+ environments, it leads to an imbalance in cellular ion concentrations and causes various negative impacts, such as impairment of photosynthesis and disruption of enzyme activities. In such situations, the intracellular Ca2+ concentration increases, triggering the activation of CBL proteins. These activated proteins bind to CIPK proteins and further activate downstream targets involved in regulating ion transport and homeostasis. Through this process, the CBL-CIPK signaling pathway can help prevent the accumulation of Na+ [60]. The extensive data obtained from this research, specifically regarding the connection between CBL genes and alkaline stress in poplar, constitutes a robust basis for future experiments.
5. Conclusions
Overall, 13 CBL genes were identified in the P. trichocarpa genome for the first time. Through multiple sequence alignment, chromosome localization, phylogenetic relationships, promoter cis-acting elements, phosphorylation site prediction, and protein interaction networks, a systematic analysis was conducted on the 13 PtrCBL genes. RNA-seq and RT-qPCR techniques were used to analyze the expression patterns and functions of the CBL family in P. simonii × P. nigra under alkaline stress. Our research provides a basis for the subsequent functional identification of the CBL genes and the study of their response to alkaline stress in poplar. More experiments will be conducted to verify the function of specific CBL genes under alkaline stress in poplar in future research.
Conceptualization, H.W., J.Y. and X.L.; methodology, J.W. and X.L.; software, H.W.; validation, Z.J. and X.L.; formal analysis, H.W.; investigation, H.W.; resources, J.W.; data curation, J.W.; writing—original draft preparation, H.W.; writing—review and editing, J.Y. and X.L.; visualization, H.W.; supervision, Z.J.; project administration, X.L.; funding acquisition, H.W. and X.L. All authors have read and agreed to the published version of the manuscript.
RNA-Seq data under alkaline stress can be found under accession number: GSE285903.
The authors declare no conflicts of interest.
Footnotes
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Enlarge this image.Figure 2. Phylogenetic tree of the CBLs in Populus trichocarpa, Arabidopsis thaliana, and Glycine max. Fonts in different colors represent different species.
Enlarge this image.Figure 3. The multiple sequence alignment of PtrCBLs in P. trichocarpa. The different EF-hand domains are marked by different colored lines. The magenta frame tagged the myristoylation domain.
Enlarge this image.Figure 4. Gene structure of PtrCBLs. (A) Phylogenetic tree within PtrCBLs in P. trichocarpa. The different background color represents the subgroup. (B) The exon–intron structure analysis of PtrCBLs. (C) The conserved motifs in PtrCBL proteins. MEME website was used to identify the conserved motifs. Modules in different colors represent different motifs.
Enlarge this image.Figure 5. Distribution of cis-elements in PtrCBL promoter regions. Different shapes represent different types of cis-elements.
Enlarge this image.Figure 7. The predicted protein interaction networks of PtrCBL proteins. (A–M) represents the interaction network of each PtrCBL protein.
Enlarge this image.Figure 8. The predicted phosphorylation sites in the PtrCBL amino acid sequence. Different colored modules represent serine, threonine, and tyrosine sites, respectively.
Enlarge this image.Figure 9. Differentially expressed genes (DEGs) in the leaves of P. simonii × P. nigra under alkaline stress. (A) DEGs statistics of RNA-seq in P. simonii × P. nigra under alkaline stress. Different colored bars represent the number of up-regulated, down-regulated, and total DEGs in the transcriptome. (B) Heatmap of the expression profiles of 13 PsnCBL genes under alkaline stress using RNA-seq data. The different colored boxes indicate different log2 (FPKM) values, with expression gradually increasing from blue to yellow to red over time. (C) The biological process at 7 d under low-concentration alkaline stress. (D) The biological process at 7 d under high-concentration alkaline stress. (E) The biological process at 14 d under low-concentration alkaline stress. (F) The biological process at 14 d under high-concentration alkaline stress.
![View Image - Figure 10. Validation of the expression data of nine PsnCBL genes from RNA-seq by RT-qPCR. The data are expressed as the mean ± standard deviation of the three biological replicates. An asterisk (*) represents a significant difference (p [less than] 0.05), and double asterisks (**) represent an extremely significant difference (p [less than] 0.01). (a) Expression analysis of PsnCBLs between RNA-seq and RT-qPCR at 7 d under different concentrations of alkaline stress. (b) Expression analysis of PsnCBLs between RNA-seq and RT-qPCR at 14 d under different concentrations of alkaline stress.](https://media.proquest.com/media/hms/THUM/20/KK8jb?_a=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%3D%3D&_s=hNf7cjqW%2Fi4tda%2BI0jnoGkxmF94%3D "View Image - Figure 10. Validation of the expression data of nine PsnCBL genes from RNA-seq by RT-qPCR. The data are expressed as the mean ± standard deviation of the three biological replicates. An asterisk (*) represents a significant difference (p [less than] 0.05), and double asterisks (**) represent an extremely significant difference (p [less than] 0.01). (a) Expression analysis of PsnCBLs between RNA-seq and RT-qPCR at 7 d under different concentrations of alkaline stress. (b) Expression analysis of PsnCBLs between RNA-seq and RT-qPCR at 14 d under different concentrations of alkaline stress.")
Enlarge this image.Figure 10. Validation of the expression data of nine PsnCBL genes from RNA-seq by RT-qPCR. The data are expressed as the mean ± standard deviation of the three biological replicates. An asterisk (*) represents a significant difference (p [less than] 0.05), and double asterisks (**) represent an extremely significant difference (p [less than] 0.01). (a) Expression analysis of PsnCBLs between RNA-seq and RT-qPCR at 7 d under different concentrations of alkaline stress. (b) Expression analysis of PsnCBLs between RNA-seq and RT-qPCR at 14 d under different concentrations of alkaline stress.
Characteristic analysis of 13 PtrCBLs in P. trichocarpa.
| Gene Name | Gene ID | Chromosome | Protein Length (aa) | Molecular Weight (kDa) | Theoretical pI | Instability Index | Aliphatic Index | Grand Average of Hydropathicity (GRAVY) | Transmembrane Domain | Predicted Localizations |
|---|---|---|---|---|---|---|---|---|---|---|
| PtrCBL1 | Potri.001G090200 | Chr01:7135427..7139480 forward | 216 | 24,588.92 | 4.64 | 38.27 | 91.16 | −0.221 | 0 | Cell membrane |
| PtrCBL2 | Potri.001G150200 | Chr01:12540135..12545031 reverse | 213 | 24,340.72 | 4.65 | 31.99 | 88.73 | −0.131 | 0 | Cell membrane |
| PtrCBL3 | Potri.001G371700 | Chr01:38923870..38928842 forward | 223 | 25,672.27 | 4.8 | 42.63 | 95.74 | −0.203 | 0 | Cell membrane |
| PtrCBL4 | Potri.003G037800 | Chr03:4115827..4119014 forward | 244 | 27,826.1 | 4.93 | 41.48 | 106.76 | −0.007 | 1 | Cell membrane |
| PtrCBL5 | Potri.003G084200 | Chr03:11086170..11091011 forward | 213 | 24,393.73 | 4.7 | 32.07 | 89.67 | −0.158 | 0 | Cell membrane |
| PtrCBL6 | Potri.003G141400 | Chr03:15725522..15727978 reverse | 226 | 25,727.34 | 4.59 | 36.48 | 94.12 | −0.130 | 0 | Cytoplasm |
| PtrCBL7 | Potri.006G002900 | Chr06:233213..238614 reverse | 226 | 25,887.34 | 4.74 | 43.90 | 89.34 | −0.290 | 0 | Cell membrane |
| PtrCBL8 | Potri.006G230200 | Chr06:23364198..23366967 reverse | 254 | 29,236.43 | 4.85 | 42.37 | 97.56 | −0.135 | 1 | Cell membrane |
| PtrCBL9 | Potri.011G094900 | Chr11:12236074..12240696 forward | 222 | 25,803.36 | 4.77 | 44.55 | 90.45 | −0.239 | 0 | Cell membrane |
| PtrCBL10 | Potri.012G015100 | Chr12:1770445..1773882 reverse | 213 | 24,498.86 | 4.78 | 45.93 | 93.29 | −0.274 | 0 | Cell membrane |
| PtrCBL11 | Potri.015G013100 | Chr15:845065..849858 forward | 213 | 24,453.94 | 4.94 | 41.35 | 91.03 | −0.305 | 0 | Cell membrane |
| PtrCBL12 | Potri.015G013200 | Chr15:851384..855678 forward | 213 | 24,009.40 | 4.69 | 39.05 | 85.96 | −0.131 | 0 | Cell membrane |
| PtrCBL13 | Potri.016G003500 | Chr16:176382..181897 reverse | 226 | 25,894.42 | 4.70 | 40.10 | 91.90 | −0.247 | 0 | Cell membrane |
Estimation of the age of duplicated PtrCBLs.
| Gene Pairs | Ka | Ks | Ka/Ks | Date (MY) | Duplication Type |
|---|---|---|---|---|---|
| PtrCBL2-PtrCBL5 | 0.021 | 0.2249 | 0.0933 | 6.81 | Purifying selection |
| PtrCBL3-PtrCBL3 | 0.01536 | 0.2044 | 0.0751 | 6.19 | Purifying selection |
| PtrCBL4-PtrCBL8 | 0.2293 | NaN | NaN | NaN | NaN |
| PtrCBL7-PtrCBL13 | 0.0247 | 0.1643 | 0.1502 | 4.97 | Purifying selection |
| PtrCBL10-PtrCBL11 | 0.0337 | 0.1645 | 0.205 | 4.98 | Purifying selection |
Supplementary Materials
The following supporting information can be downloaded at:
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; Wu, Juan 1 ; Ju, Zhixin 1 ; Yang, Jingli 2 ; Leng, Xue 1 1 College of Agriculture, Jilin Agricultural Science and Technology University, Jilin 132101, China;
2 State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China