Next Article in Journal
Post-Fire Recovery of Soil Multiple Properties, Plant Diversity, and Community Structure of Boreal Forests in China
Previous Article in Journal
The Hidden Carbon Cost of Forest Fire Management: Quantifying Greenhouse Gas Emissions from Both Vegetation Burning and Social Rescue Activities in Yajiang County, China
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Forests
Volume 16
Issue 5
10.3390/f16050805
forests-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

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

Genome-Wide Identification, Evolution and Expression Analysis of the G-Protein Gene Family in Poplar (Populus alba × Populus glandulosa)

by
Bobo Song
2,†,
Qian Liu
1,†,
Zitong Zeng
1,
Yiyang Gu
1,
Wenxin Ye
1,
Fangfang Fu
1 and
Meiling Ming
1,*
1
State Key Laboratory of Tree Genetics and Breeding, Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
2
College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2025, 16(5), 805; https://doi.org/10.3390/f16050805
Submission received: 22 March 2025 / Revised: 22 April 2025 / Accepted: 9 May 2025 / Published: 12 May 2025
(This article belongs to the Section Genetics and Molecular Biology)
Browse Figures
Versions Notes

Abstract

:
Heterotrimeric G-proteins are key signal transduction mediators involved in regulating plant growth and development, and responses to various stress in plants. G-proteins are extensively investigated in model plants, such as Arabidopsis thaliana and Oryza sativa. However, the identification and function of G-proteins in woody species, particularly Populus, remain largely unexplored. In this study, we performed a genome-wide identification and comprehensive analysis of the G-protein gene family in poplar, aiming to reveal their evolutionary history, structural diversity and potential function roles. As a result, a total of 27 G-protein genes were identified in the poplar genome, including 18 Gα, 4 Gβ and 5 Gγ subunits. Phylogenetic analysis indicated that 27 G-protein genes were divided into three subgroups. Gene structure, conserved domain and motifs indicated the conserved nature of G-protein at sequence and structure. In addition, synteny analysis revealed that whole-genome duplication events contributing to the expansion of the G-protein gene family in poplar. Cis-regulatory element analysis indicated that many G-protein genes in poplar contain hormone and stress related motifs, suggesting that G-protein genes are involved in environmental adaptation. Expression profiling analysis demonstrated that G-protein genes exhibited tissue-specific expression and stress-responsive expression patterns, highlighting their potential regulatory roles in growth and development and responses to biotic and abiotic stresses. This study provides valuable insights into the poplar G-protein gene family and lays the foundation for further functional analyses, contributing to improving stress tolerance in forestry species.

1. Introduction

Heterotrimeric G-proteins are signal transduction proteins widely present in eukaryotic organisms [1]. Heterotrimeric G-protein consists of three subunits, including Gα, Gβ and Gγ. Heterotrimeric G-proteins play crucial roles in regulating various physiological processes, such as hormone signal transduction, cell proliferation and stress response [2,3]. In plants, G-proteins were first studied in the model plant Arabidopsis thaliana. Unlike in plants, human G-protein exhibited extensive diversity, including 23 Gα, five Gβ and 13 Gγ subunits [4,5,6]. Initially, only a small number of G-protein subunits were identified in Arabidopsis, including only one Gα subunit, one Gβ subunit and two Gγ subunits [7,8,9]. Recently, plant-specific atypical G-proteins have been identified, further expanding the knowledge of this field. First, extra-large Gα subunits (XLGs) were identified in many plants and these XLGs can interact with Gβ and Gγ subunits to form trimers that function similar to typical Gα subunits [10,11]. Another important finding was the identification of various types of Gγ in plants [12]. These findings provide a structural foundation for the formation of diverse assemblies of plant heterotrimer G-proteins and their participation in the regulation of various physiological reactions.
The Gα subunit function as a GTPase, binding and hydrolyzing GTP [13]. Upon GTP hydrolysis to GDP, it reassociates with the Gβγ dimer to form a heterotrimeric complex. Gα contains a typical RAS domain and a helical domain, which regulate intracellular signal transduction cascades. In plants, Gα subunits generally exhibit high intrinsic activation ability, meaning they can be activated without requiring a G-protein-coupled receptor (GPCR) to facilitate GTP binding [13]. The Gβ subunit is a WD40 repeat protein that typically forms a dimer with the Gγ subunit. Upon Gα release, this Gβγ dimer can function independently in signal transduction. In plants, Gβ subunit diversity is relatively low. For example, Arabidopsis contains only a single Gβ subunit (AGB1), yet it is widely distributed across the plasma membrane, internal membranes and multiple tissues [14,15]. The Gγ subunit forms a dimer with Gβ and reassociates with Gα after GTP hydrolysis, thereby completing the G-protein signaling cycle. Based on C-terminal modifications, Gγ subunits can be classified into type I, type II and type III. Type I Gγ proteins are typically prenylated for membrane localization, whereas type III Gγ proteins contain multiple cysteine residues at the C-terminus, allowing them to interact with XLG proteins [16].
In the plant kingdom, G-proteins play critical roles in the growth and development of vegetative organs, regulation of stomatal development and movement, reproductive growth and embryonic development, responses to biotic and abiotic stresses, and nodule formation. For example, in tomato, deletion of a type II Gγ gene alters plant sensitivity to auxin and ABA, resulting in smaller seeds and changes in fruit shape [17]. Several studies have demonstrated that heterotrimeric G-protein signaling components are involved in plants’ cellular immune responses. In Arabidopsis, XLG2 and Gβ function together in defense against Pseudomonas syringae, enhancing resistance to this pathogen [18]. During plant responses to low temperature, high temperature, drought and salt stress, various heterotrimeric G-protein signaling components contribute to the induction of stress tolerance. The expression levels of GPCR genes, such as COLD1 in rice and maize and ShGPCR1 in sugarcane, are upregulated under low temperature, drought, salt and cold stress, promoting the activation of resistance genes and enhancing plant stress tolerance [19,20]. In wheat, Gβ gene knockout mutants exhibit reduced drought tolerance, whereas Gβ-overexpressing lines show enhanced drought resistance. Under drought and salt stress, wheat cells in Gβ-overexpressing lines display higher superoxide dismutase (SOD) activity, lower levels of membrane lipid peroxidation products (MDA) and increased proline accumulation, thereby improving cellular tolerance to environmental stress [21].
Poplar (Populus alba × Populus glandulosa) is widely studied in forestry research due to its adaptability, rapid growth, high biomass production and efficient propagation [22,23,24,25]. The poplar genome is relatively small (P. alba [subgenome A]: 356 Mb; P. tremula var. glandulosa [subgenome G]: 354 Mb) and enables genetic manipulation, making it an ideal model species for studying tree molecular biology. G-protein have been identified in many species, such as barley [26], wheat [27] and soybean [28]. However, studies on G-proteins in poplar remain limited. These proteins are crucial for various cellular signaling pathways and play a significant role in the regulation of plant growth, development and stress responses. Research on G-proteins in poplar could pave the way for enhancing stress tolerance and advancing poplar breeding programs. This study focuses on the G-protein gene family in poplar, aiming to characterize its evolutionary relationships, gene structure and potential functional roles in stress adaptation. In this study, we performed a whole genome identification of the G-protein gene family in poplar. We conducted physicochemical property analysis, phylogenetic tree analysis, chromosomal localization analysis, gene structure and conserved motif analysis. Furthermore, collinearity analysis and duplication event analyses were carried out to elucidate the evolutionary history of the G-protein gene family in poplar. Additionally, transcriptome data analysis was performed to uncover the expression patterns of G-protein genes across different tissues and under various stress conditions. Our findings provide new insights into the potential roles of poplar G-proteins in responses to biotic and abiotic stress, contributing to future functional studies and molecular breeding efforts in poplar and other forest trees.

2. Results

2.1. Genome-Wide Identification of G-Protein Gene Families in Poplar

In our study, a total of 687, 22 and 11 candidate hits were identified for Gα, Gβ and Gγ protein, respectively. The overlapping members identified by both the BLAST (v2.13.0+) and HMMsearch methods were obtained. Then, we removed the member with the incomplete G-protein domains based on the CDD website. Finally, a total of 27 G-proteins were identified in the poplar genome. Based on the difference of the three conserved domains (Gα, Gβ and Gγ), we categorized the identified G-proteins. In the poplar genome, there are 18 Gα, four Gβ and five Gγ proteins (Table 1). To distinguish different G-protein member, we named these genes based on their chromosomal order within each class (PagGα1-PagGα18, PagGβ1-PagGβ4 and PagGγ1-PagGγ5), (Table 1). In subgenome A (P. alba), we identified nine Gα proteins, two Gβ proteins and two Gγ proteins. In subgenome G (P. tremula var. glandulosa) we identified nine Gα proteins, two Gβ proteins and three Gγ proteins. Compared to subgenome A, subgenome G contains an additional PagGγ3 gene.
The ExPASy Proteomics Server, an online proteomics and sequence analysis tool, was used to predict the physicochemical properties of G-proteins in poplar. Among these 27 identified G-proteins, PagGα14 had the longest length of protein sequence (926 amino acids), whereas PagGγ1 and PagGγ4 had the shortest length of amino acid sequence (110 amino acids). The theoretical isoelectric points (pI) of these G-proteins ranged from 4.91 (PagGγ1) to 9.75 (PagGγ5); the molecular weight of G-proteins ranged from 12,408.97 (PagGγ1) to 103,154.12 (PagGα6) kDa. In addition, their grand average of hydropathicity indices ranged from −0.701 to 0.07. Meanwhile, we also investigated the instability index and aliphatic index of poplar G-proteins. The prediction of subcellular localization indicated that 20 G-proteins were located in the nuclear, three G-proteins in the chloroplast, three G-proteins in the cytoplasmic, and one in the mitochondrial region. These results indicated that G-proteins might exhibit diverse functional roles in intracellular signaling and cellular compartmentalization (Table 1).

2.2. Chromosomal Localization Analysis of G-Protein Genes in the Poplar Genome

To explore the chromosomal distribution pattern of G-protein genes, 27 G-protein genes were mapped onto the poplar genome. We used the TBtools software (v2.210) to display the distribution of G-protein genes. As a result, we observed an uneven distribution of 27 G-protein genes across poplar chromosomes. While poplar genome consists of 19 chromosomes, G-protein genes were mapped to only 10 chromosomes in both subgenome A and subgenome G, including chrA01, chr03, chr04, chr05, chr06, chr07, chr09, chr12, chr15 and chr18. In subgenome A, chrA01, chrA03, chrA04, chrA05, chrA06, chrA07 and chrA12 each only contained one G-protein gene; for subgenome G, chrG01, chrG03, chrG04, chrG05, chrG07 and chrG12 each only contained one G-protein gene. In subgenome A, chrA09, chrA15 and chrA18 contained two G-protein genes; in subgenome G, chrG06, chrG09, chrG15 and chrG18 contained two G-proteins. Interestingly, chrG06 in subgenome G harbored one additional Gγ protein gene (PagGγ3) compared to subgenome A. This result suggests the differences in genome evolution between two subgenomes (Figure 1).

2.3. Phylogenetic Analysis of G-Protein in Poplar

To reveal the evolutionary relationship of the identified G-protein in poplar, we constructed a Neighbor-Joining (NJ) phylogenetic tree using MEGA software (v10.2.2) using the protein sequences of 27 G-proteins in poplar and eight G-proteins in Arabidopsis (Figure 2). Subsequently, these G-proteins were phylogenetically categorized into three subgroups, corresponding to the Gα, Gβ and Gγ classes. In Arabidopsis, four, three and one G-protein(s) were clustered into Gα, Gβ and Gγ subgroups, respectively; in rice (Oryza sativa), five, one and five G-protein(s) were clustered into Gα, Gβ and Gγ subgroups, respectively; in poplar, eighteen, five and four G-proteins were clustered into Gα, Gβ and Gγ subgroups, respectively. This result was consistent with the classification based on G-protein domain analysis. Notably, the Gα subgroup contained a greater proportion of G-proteins than the Gβ and Gγ subgroups. It is important to note that most sister pairs of subclades consisted of Arabidopsis G-protein and poplar G-protein (such as, AtAGB1 and PagGβ1, PagGβ2, PagGβ3, PagGβ4), suggesting that these G-protein might have conserved function. However, some G-proteins, such as PagGα7, PagGα8, PagGα16 and PagGα17, were phylogenetically distinct from both Arabidopsis and other poplar G-proteins. This result suggested that part of the G-protein might have undergone functional differentiation during the evolution process in Arabidopsis and poplar.

2.4. Gene Structure, Conserved Domain and Motif Analysis of G-Proteins

To further understand the characteristics of G-proteins in poplar, we analyzed the gene structure, conserved domains and motif composition of the G-protein gene family. The clustering was based on the phylogenetic analysis of G-proteins in poplar (Figure 3A). As a result, the 27 G-proteins were classified into three subgroups corresponding to Gα, Gβ and Gγ proteins.
In this study, we identified 20 conserved motifs in G-protein gene family in poplar using MEME website tool (Figure 3B). To order to distinguish these motifs, we named them as Motif 1-Motif 20 (Figure S1). The conserved motif analysis revealed that members of the same subgroup exhibited similar motif compositions, whereas different subgroups displayed distinct motif patterns. For example, in the Gα protein subgroup, all Gα proteins in poplar contained Motif 2, Motif 4, Motif 5, Motif 7, Motif 9, Motif 10 and Motif 12. Motif 15, Motif 16 and Motif 20 were specifically detected in the Gβ protein subgroup. In the Gγ protein subgroup, except for PagGγ2, all Gγ proteins contained only Motif 12. This result suggests that the function of PagGγ2 may have diverged or been lost. These variations within the same subgroup indicate potential functional diversity among genes of the same class.
Gene structure is an important feature that might influence gene expression and function. Here, we analyzed the exon-intron compositions of the 27 G-protein genes (Figure 3C). The number of exons (CDSs) ranged from 4 (e.g., PagGγ2) to 14 (PagGα18). The number of introns ranged from 4 to 14. In addition, 15 genes contained 5′UTR and 11 genes exhibited 3′UTR. These differences in gene structure indicate the functional diversity of G-protein in poplar. In addition, the G-proteins clustered within the same subclades exhibited similar gene structure characteristics. We also detected the conserved domain of G-protein gene family members. A total of five types of conserved domain were detected, including G-alpha, FYVE_linke_SF superfamily, G-gamma, G-gamma superfamily, PTZ00169 superfamily and WD40. All genes in the Gα protein subgroup contained the G-alpha domain; all genes in the Gβ protein subgroup contained the WD40 domain; all genes in the Gγ protein subgroup contained the G-gamma or G-gamma superfamily domain (Figure 3D). These findings provide evidence to support the results of the phylogenetic classification based on the homology of Gα(G-alpha), Gβ(WD40) and Gγ(G-gamma) proteins.

2.5. Cis-Acting Elements Analysis in the Promoter of G-Proteins in Poplar

Generally, the cis-acting elements in the promoter of genes play a crucial role in regulating gene expression and influencing gene function. In this study, we extracted the putative promoter regions (2 kb upstream of the transcription initiation site) of all G-protein gene family members and then submitted to the PlantCARE website to predict the cis-acting elements. A total of 88 classes of cis-acting element were identified in poplar G-protein genes and 18 important cis-acting elements were selected for further analysis (Table S1). In Figure 4A, a diversity of cis-elements distribution was observed, suggesting that G-protein can be regulated by different factor or environment, such as, light, gibberellin, low-temperature, MeJA, abscisic acid, defense and stress etc. Among all cis-elements, light responsiveness was most frequently found in the poplar G-protein gene promotors (Figure 4B). The cis-acting elements involved in defense and stress responsiveness were predicted on the putative promotor of 12 G-protein genes; these results highlighted the important role of G-protein in stress responsiveness. For hormone-related elements, cis-acting element involved in the abscisic acid responsiveness was most frequently found in the poplar G-protein gene promotors. Abscisic acid (ABA) is a key plant hormone that plays a vital role in mediating responses to diverse stress signals and these cis-acting element on the poplar. In addition, eight G-protein genes contained cis-elements involved in auxin responsiveness, suggesting that G-protein plays an important role in the auxin signaling pathway [17].
G-protein gene promotors contribute to their functional diversity under stress conditions. In summary, the cis-acting analysis revealed that G-protein genes play important roles in stress responsiveness in poplar.

2.6. Collinearity Analysis of G-Protein Gene Family in Poplar

To order to investigate the expansion and evolution history of the G-protein gene family in poplar, we performed a collinearity analysis using two subgenomes (subgenome A and subgenome G). For subgenome A (P. alba), 18,739 (43.9%) genes were produced by whole genome duplication (WGD) or segmental duplication; 9931 (23.3%) genes were produced by singleton duplication; 8247 (19.3%) genes were produced by dispersed duplication; 2726 (6.4%) genes were produced by proximal duplication; and 3036 (7.1%) genes were produced by tandem duplication. For subgenome G (P. tremula var. glandulosa), 17,893 (42.2%) genes were produced by WGD or segmental duplication; 9520 (22.4%) genes were produced by singleton duplication; 8735 (20.6%) genes were produced by dispersed duplication; 3045 (7.2%) genes were produced by proximal duplication; and 3234 (7.6%) genes were produced by tandem duplication (Figure 5A). Regarding the G-protein gene family, for subgenome A, eight G-protein genes were produced by WGD or segmental duplication; four and one G-protein genes were produced by dispersed and proximal duplication, respectively. For subgenome G, 13 genes were produced by WGD or segmental duplication, while only one gene were produced by dispersed duplication (Figure 5B and Table S2). These results revealed that WGD or segmental duplication events were the major forces in the expansion of the G-protein gene family.
To further investigate the evolutionary patterns of G-protein genes in poplar genome, we used MCScanX (v1.0.0) software to identify the synteny blocks and synteny gene pairs in the poplar genome. As a result, a total of 476 synteny blocks (8680 synteny gene pairs) were identified in subgenome A and 456 synteny blocks (11,968) were identified in subgenome G (Figure 5C). Among the G-protein gene family, four synteny gene pairs (five genes, PagGα3-PagGα5, PagGα3-PagGα6, PagGα5-PagGα6, PagGα7-PagGα8) were identified in subgenome A; eight synteny gene pairs (13 genes, PagGα10-PagGα11, PagGβ3-PagGβ4, PagGα12-PagGα14, PagGα12-PagGα15, PagGα13-PagGα18, PagGγ3-PagGγ5, PagGα14-PagGα15, PagGα16-PagGα17) were identified in subgenome G (Table 2).

2.7. Expression Patterns of G-Protein Genes in Different Tissues of Poplar

In this study, we performed transcriptome sequencing analysis to investigate the expression patterns of G-protein genes in different tissues of poplar. Four different tissues (root, leaf, bark and xylem) of ‘P. tremula × P. alba’ and four different tissues (shoot, root, leaf and callus) of ‘P. alba × P. tremula var. glandulosa’ were collocated from a previous study [29,30,31,32]. We performed RNA-seq analysis and quantified the gene expression using Fragments Per Kilobase of exon per Million (FPKM) values. In Figure 6, the expression patterns of all G-protein genes are shown. For ‘P. alba × P. tremula var. glandulosa’, 6, 7, 5 and 9 genes exhibited the highest expression level in shoot, root, leaf and callus, respectively. For ‘P. tremula × P. alba’, 17, 1, 7 and 2 genes exhibited the highest expression level in root, leaf, bark and xylem, respectively. These results suggest that G-protein genes play diverse roles in poplar tissue development (Tables S3 and S4). Interestingly, eight genes (PagGα13, PagGα7, PagGα16, PagGα8, PagGα15, PagGγ1, PagGγ4 and PagGα18) were highly expressed in callus of ‘P. alba × P. tremula var. glandulosa’. A previous study had reported that G-protein are involved in cell proliferation of Arabidopsis [33]. Therefore, these genes may be associated with the cell division and cell proliferation during callus growth and development process. In addition, 17 genes (PagGα2, PagGα11, PagGα14, PagGα15, PagGβ3, PagGα10, PagGα5, PagGγ1, PagGα8, PagGα17, PagGα3, PagGγ5, PagGγ2, PagGα12, PagGβ1, PagGα7, PagGα16) exhibited high expression levels in the root of ‘P. tremula × P. alba’. Therefore, the high expression of these 17 genes in the roots of ‘P. tremula × P. alba’ suggests their potential involvement in stress responses, as roots play a crucial role in absorbing water and nutrients, as well as in responding to environmental stresses such as drought, salinity and soil pathogens (Figure 6).

2.8. Expression Patterns of G-Protein Genes Under Biotic and Abiotic Stresses

The expression pattern can provide important clues for predicting gene function. To explore the potential function of G-protein gene family in biotic and abiotic stresses responsiveness, we downloaded transcriptome data related to biotic (Alternaria alternata infection) and abiotic stresses (salt, drought, high temperature, and low temperature) from public databases [34].
First, we detected the expression pattern of G-protein gene family under pathogens treatment (A. alternata-treated leaves), a type of biotic stress (Table S5). Several G-protein genes exhibited significant induction at different time points following pathogen treatment. Specifically, PagGα5, PagGα7, PagGα16, PagGα18 and PagGα11 were highly induced after 2 days of pathogen treatment, while PagGα8 and PagGα17 were highly induced after 3 days. Additionally, PagGα1, PagGα2 and PagGα10 were strongly upregulated after 4 days of pathogen exposure (Figure 7A). These results suggest that these G-protein genes may play crucial roles in pathogen defense responses.
Next, we investigated the expression patterns of G-protein genes under various abiotic stresses, including salt, drought, high temperature and low temperature (Table S6). The results revealed distinct expression patterns of G-protein genes under different stress conditions. Specifically, PagGα9, PagGβ1, PagGβ3 and PagGγ5 were highly expressed under cold stress, while PagGα6, PagGα15, PagGγ1 and PagGα4 exhibited high expression levels under heat stress. Additionally, PagGα16 showed relatively higher expression levels under drought stress. However, G-protein gene expression was generally lower under salt stress, regardless of short-term or prolonged exposure (Figure 7B). These findings suggest that G-protein genes may have undergone functional differentiation, as they exhibit distinct expression patterns in response to different abiotic stress conditions.

3. Discussion

G-proteins are important for regulating the growth and development of vegetative organs, facilitating reproductive processes and embryonic development and mediating responses to both biotic and abiotic stressors [17,18,19,20]. In this study, we identified several key G-protein genes in poplar, analyzing their structure, phylogenetic relationships and expression patterns. We observed that G-protein genes in poplar display distinct tissue-specific expression profiles, with significant upregulation in response to biotic and abiotic stressors.
AtGPA1, a G-protein subunit gene, was first reported in Arabidopsis in 1990 [35]. Subsequently, an increasing number of G-protein subunits have been cloned in the plant kingdom. In animals, G-protein subunits exhibit extensive diversity, contributing to the broad function roles of G-proteins in many different signal pathways. However, in plants, the diversity of G-protein subunits is relatively limited. To date, a total of one Gα subunit gene (AtGPA1), three XLG genes (AtXLG1, AtXLG2, AtXLG3), one Gβ gene (AtAGB1) and three Gγ genes (AtAGG1, AtAGG2, AtAGG3) have been identified in Arabidopsis [12]. In the rice genome, there are one Gα subunit gene (OsRGA1), four XLG genes (OsXLG1, OsXLG2, OsXLG3, OsXLG4), one Gβ gene (OsRGB1) and five Gγ subunits (OsRGG1, OsDEP1, OsGS3, OsGGC2) [10,36,37]. In our study, similarly, we detected nine Gα proteins, two Gβ proteins and two Gγ proteins in subgenome A (P. alba). In subgenome G (P. tremula var. glandulosa), we identified nine Gα proteins, two Gβ proteins and three Gγ proteins. Compared to subgenome A, subgenome G contains one additional PagGγ3 gene. The relatively higher number of Gα and Gβ proteins in poplar may be attributed to whole-genome duplication (WGD) or segmental duplication events. Previous studies had reported that all poplar species had undergone a recent WGD event before species diversification [38].
Based on the characteristics of conserved domains, G-proteins are classified into three main classes: Gα protein, Gβ protein and Gγ protein [39]. In our study, phylogenetic analysis, gene structure analysis, conserved domain and motif analysis revealed that the G-protein gene family in poplar can be divided into three classes. The G-protein members in the same class exhibited similar gene structures, conserved domains and motifs. These findings suggested that the sequences of G-proteins are conserved. Among them, Gα class comprises the largest number of genes within the poplar G-protein family. Based on the clustered results of phylogenetic analysis, we can further divide Gα protein in poplar into two small subgroups, extra-large Gα subunits (XLGs) and typical Gα subunits (GPAs). There are 14 Gα subunits (PagGα7, PagGα16, PagGα8, PagGα17, PagGα11, PagGα2, PagGα1, PagGα10, PagGα6, PagGα12, PagGα15, PagGα3, PagGα5, PagGα14) in poplar that may be the candidate XLG genes; four Gα subunits (PagGα4, PagGα9, PagGα13, PagGα18) may be candidate GPA genes. XLG proteins generally have longer sequences than typical Gα subunits, with lengths approximately twice that of Gα proteins [26]. In our study, 14 XLG subunits (average value: 881 amino acids) had a longer protein sequence than four GPAs (average value: 397 amino acids). This difference in sequence length may be due to the fact that N-terminal of XLG subunits contain an elongated region consisting of 300 to 500 amino acids [1].
Heterotrimeric G-proteins play important roles in plant signaling transduction, mediating plant growth and development, and responses to biotic and abiotic environmental stimuli [40]. Our study highlights the diverse regulatory functions of G-proteins in poplar, a widely distributed genus known for its remarkable adaptability to different ecological conditions. Through cis-acting element and gene expression analyses, we have identified key G-protein genes that are differentially expressed across tissues and in response to various biotic and abiotic stresses. The identification of specific cis-regulatory elements in the promoter regions of G-protein genes suggests that G-protein genes in poplar involvement in hormone signaling pathways such as auxin, abscisic acid (ABA) and gibberellin, which are critical for plant growth and environmental adaptation. Notably, genes with ABA-responsive elements were highly expressed under drought treatment, indicating a potential role in stress resistance. For example, PagGα16 harbored a cis-acting element involved in ABA responsiveness in its promoter region (Figure 4) and was highly expressed under drought treatment (Figure 7). These findings are consistent with previous research in Arabidopsis and rice, where G-proteins are involved in various stress responses, particularly in regulating stomatal movement and enhancing drought tolerance. In tomato, deletion of a type II Gγ gene alters plant sensitivity to auxin and ABA, resulting in smaller seeds and changes in fruit shape [17]. Similarly, the enrichment of auxin-responsive elements in the promoters of G-protein genes further supports the role of G-proteins in plant growth and morphogenesis.
The tissue-specific expression patterns of G-protein genes in poplar are consistent with poplar’s broad adaptability to various environments. The high expression of genes in roots may contribute to stress tolerance. For example, 17 genes (e.g., PagGα2, PagGα11 and PagGα14) exhibited highly expression in root of ‘P. tremula × P. alba’. In contrast, five G-protein genes (e.g., PagGα11, PagGα12 and PagGα14) were expressed predominantly in leaves of ‘P. alba × P. tremula var. glandulosa’, indicating that these G-protein genes may regulate photosynthetic efficiency and stomatal regulation, optimizing water use and gas exchange under fluctuating environmental conditions [41]. Moreover, G-proteins also play a role in plant immune responses, as evidenced by their upregulation under pathogen attack [42]. Expression pattern analysis of G-protein revealed that some genes (e.g., PagGα5, PagGα7, PagGα18) were induced expression after pathogen treatment, indicating that these G-protein genes might involve in pathogen stress. This result is consistent with previous reports in Arabidopsis that G-protein (XLG2 and Gβ) is involved in defense against Pseudomonas syringae, enhancing resistance to this pathogen [18].
In conclusion, our comprehensive analysis highlights the critical role of G-proteins in regulating plant development and stress adaptation. The functional diversity of G-protein genes in Populus likely contributes to its ecological success, reinforcing its potential as a model for studying environmental resilience in woody plants.

4. Materials and Methods

4.1. Whole Genome Identification of G-Protein Gene Family Member in Poplar

First, we downloaded the poplar (Populus alba × Populus tremula var. glandulosa clone 84K) genome data from National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/, accessed on 3 March 2025) [23], including chromosome data, whole genome coding sequences (cds), whole genome protein sequences (pep) and annotation files (gff3). Then, BLAST (v2.13.0+) and HMMsearch (v3.4) software were performed to identify G-protein members in poplar [43,44]. In our study, two methods were used to construct a whole genome protein database of poplar using whole genome protein sequences of poplar. The amino acid sequences of G-protein gene family members in Arabidopsis were downloaded from The Arabidopsis Information Resource (TAIR) database (https://v2.arabidopsis.org/, accessed on 3 March 2025). The amino acid sequences of G-protein gene family members in rice were downloaded from NCBI. Using Arabidopsis G-protein gene family members as queries, we scanned the whole genome protein database of poplar using BLASTP (v2.13.0+) software in BLAST (E-value < 1 × 10−5, Identity > 30%) and obtained candidate G-protein members in poplar. Meanwhile, we obtained the Hidden Markov models (HMMs) of the G-protein domains (Gα [PF00503], Gβ [PF00400] and Gγ [PF00631]) from the Pfam database (http://pfam.xfam.org/, accessed on 3 March 2025). HMMsearch (v3.4) software was used to search the whole genome protein sequences of poplar based on three HMM profiles with the G-protein domains (E-value < 1 × 10−5). Redundant sequences between BLAST and HMMsearch analyses were removed (Figure S2). The Conserved Domain Database (CDD) website tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 3 March 2025) was used to detect the completeness of the G-protein domains one by one (E-value < 0.01).

4.2. The Physicochemical Properties Analysis of G-Proteins

We evaluated the physicochemical properties of G-proteins, including amino acid number, molecular weight, theoretical isoelectric point (pI), instability index, aliphatic index, grand average of hydropathicity using ExPASy ProtParam website (https://web.expasy.org/protparam/, accessed on 3 March 2025). The subcellular localization was predicted for each G-protein member using the online tool WoLF PSORT (https://wolfpsort.hgc.jp/, accessed on 3 March 2025). The distribution of identified G-protein genes across chromosomes were extracted from the poplar genome annotation file using an in-house Python (v 3.9.13) script and TBtools (v2.210) software was used for visualization [45].

4.3. Phylogenetic Analysis

The protein sequences of all G-proteins in poplar were extracted from whole-genome protein sequences (pep) of poplar. The protein sequences of all G-proteins in Arabidopsis were downloaded from the TAIR database. We used ClustalW (v2.1) software (default parameters) to perform multiple sequence alignment of G-proteins in Arabidopsis and poplar [46]. Then, MEGA (v10.2.2) software was used to construct a Neighbor-Joining (NJ) phylogenetic tree [47]. The bootstraps value was set to 1000. The evolview (version 2) website (https://www.evolgenius.info/evolview-v2/, accessed on 3 March 2025) was used to visualize this NJ phylogenetic tree.

4.4. Gene Structure, Conserved Domain and Motif Analysis

Gene structure information of G-proteins in poplar was extracted from the gene annotation file (gff file) from poplar using our in-house Python scripts. The location information of UTR, CDS and intron was extracted. TBtools was used to display the distribution of UTR, CDS and introns on G-protein gene. We used the MEME website (http://meme-suite.org/tools/meme, accessed on 3 March 2025) to identify the conserved motif element on G-proteins in poplar. The parameters were set as follows: repetitions, any number; number of different motifs, 20; minimum motif width, 30; maximum motif width, 70. TBtools (v2.210) was used to display the distribution of conserved motif on G-proteins in poplar. We used CDD website tools to identify the conserved domain of G-proteins in poplar (E-value < 0.001). TBtools (v2.210) were used to display the distribution of conserved domain on G-proteins.

4.5. Cis-Elements Analysis

The promoter sequences (2k upstream region of the transcription initiation site) of G-protein genes were extracted from the poplar genome using our in-house Python script. Then, PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 3 March 2025) was used to predict cis-acting elements. TBtools software was used to visualize the distribution of cis-acting elements on the promoter of G-protein genes.

4.6. Collinearity Analysis of G-Protein Genes

The collinearity analysis was performed following the guidelines provided by DupGen_finder (https://github.com/qiao-xin/DupGen_finder, accessed on 3 March 2025) [48]. In our study, we performed collinearity analysis in subgenome A and subgenome G. First, we used makeblastdb software to construct index files using the whole genome protein sequence of poplar genome [43]. Then, a BLASTP alignment was conducted to identify candidate homologous gene pairs using whole genome protein sequence as a query (E-value < 1 × 10−10, max_target_seqs = 5). The duplicate_gene_classifier software was used to determine duplication events. Five duplication events include whole genome duplication or segmental duplication, singleton duplication, dispersed duplication, proximal duplication and tandem duplication. Collinearity blocks and collinearity gene pairs were identified using MCScanX software with default parameters [49]. The collinearity information and gene/transposable element (TE) density on poplar genome were displayed by plotting circle pictures using circos software (version 0.69.2).

4.7. Transcriptome Sequencing Analysis

The transcriptome data were downloaded from National Center for Biotechnology Information database (NCBI, https://www.ncbi.nlm.nih.gov/). Based on previous studies, we obtained the RNA-seq data of ‘P. tremula × P. alba’ at four different tissues: root, leaf, bark and xylem and RNA-seq data of ‘P. alba × P. tremula var. glandulosa’ at four different tissues, including shoot, root, leaf and callus [29,30,31,32]. We also collected the transcriptome data of abiotic treatment (salt treatment, drought treatment, high and low temperature treatment) and pathogen treatments (A.alternata impregnated leaves) from previous study (accession number: ERR1864411-ERR1864437 and SRR12371687-SRR12371698) [34]. Ascp (v3.5.6) software (https://www.ibm.com/products/aspera, accessed on 3 March 2025) was used to download these raw data from NCBI, then fastQC (v0.12.1) software (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 3 March 2025) was used to evaluate the quality of raw data. Trimmomatic (v0.39) software was used to remove adaptor sequences and low-quality reads (Parameters: LEADING:30 HEADCROP:10 TRAILING:20 SLIDINGWINDOW:3:20 MINLEN:20) [50]. The high-quality reads were mapped to the ‘P. alba × P. tremula var. glandulosa’ genome using HISAT2 (v2.2.1) software [51]. Samtools (v1.14) software was used to convert file format (sam to bam) and sort the bam file [52]. We used featureCounts (v2.0.6) software to quantify the reads mapped on genes [53] and used Fragments Per Kilobase per Million mapped reads (FPKMs) to calculate gene expression level. The heatmaps were plotted using the pheatmap package in R (v4.0.2) [54].

5. Conclusions

In our study, we performed a whole genome identification of the G-protein gene family in poplar. A total of 27 G-proteins were identified. Through phylogenetic tree analysis, gene structure, conserved motif and domain analysis, 27 G-proteins were classified into three subfamilies: Gα, Gβ and Gγ. Collinearity and duplication analysis revealed that whole-genome duplication was the majority driver force of G-protein family expansion in poplar. Cis-acting element analysis revealed the enrichment of stress- and hormone-responsive motifs in the promoter regions, implying a key regulatory role under stress conditions. Moreover, transcriptome data demonstrated that G-protein genes exhibit diverse tissue-specific and stress-responsive expression patterns, suggesting their important roles in plant growth, development and stress adaptation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f16050805/s1, Figure S1: Amino acid compositions of 20 conserved motifs of G-protein gene family in poplar. Figure S2: Workflow for identifying G-protein genes in poplar. Table S1: Cis-acting elements analysis in the promoter of G-proteins in poplar. Table S2: Gene duplication analysis of G-protein gene family. Table S3: The expression level of 27 G-protein genes across different tissues in ‘P. tremula × P. alba’. Table S4: The expression level of 27 G-protein genes across different tissues in ‘P. alba × P. tremula var. glandulosa’. Table S5: Expression profiles of G-protein genes under pathogen (A. alternata) treatment. Table S6: Expression profiles of G-protein genes under four abiotic treatments, including drought, salt, high temperature and low temperature.

Author Contributions

Conceptualization, M.M.; data curation, Q.L., Z.Z., Y.G., W.Y. and F.F.; formal analysis, B.S., M.M. and Q.L.; writing—original draft preparation, B.S. and Q.L.; writing—review and editing, M.M., B.S. and Q.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Biological Breeding-National Science and Technology Major Projects (2023ZD0405601), the National Natural Science Foundation of China (32301613) and the China Postdoctoral Science Foundation (2023M741721).

Data Availability Statement

The transcriptome data were download from NCBI (accession number: ERR1864411-ERR1864437 and SRR12371687-SRR12371698).

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
pITheoretical isoelectric points
WGDWhole genome duplication
HMMHidden Markov model
TETransposable element
CDSCoding sequence
ABAAbscisic acid
FPKMsFragments Per Kilobase of exon per Million mapped reads

References

  1. Maruta, N.; Trusov, Y.; Jones, A.M.; Botella, J.R. Heterotrimeric G Proteins in Plants: Canonical and Atypical Gα Subunits. Int. J. Mol. Sci. 2021, 22, 11841. [Google Scholar] [CrossRef] [PubMed]
  2. Anantharaman, V.; Abhiman, S.; de Souza, R.F.; Aravind, L. Comparative genomics uncovers novel structural and functional features of the heterotrimeric GTPase signaling system. Gene 2011, 475, 63–78. [Google Scholar] [CrossRef]
  3. Fan, M.; Li, J.; Zhang, T.; Huo, H.; Lü, S.; He, Z.; Wang, X.; Zhang, J. Genome-wide identification of heterotrimeric G protein genes in castor (Ricinus communis L.) and expression patterns under salt stress. BMC Genom. 2024, 25, 1115. [Google Scholar] [CrossRef]
  4. McCudden, C.; Hains, M.; Kimple, R.; Siderovski, D.; Willard, F. G-protein signaling: Back to the future. Cell. Mol. Life Sci. 2005, 62, 551–577. [Google Scholar] [CrossRef] [PubMed]
  5. Hurowitz, E.H.; Melnyk, J.M.; Chen, Y.-J.; Kouros-Mehr, H.; Simon, M.I.; Shizuya, H. Genomic characterization of the human heterotrimeric G protein α, β, and γ subunit genes. DNA Res. 2000, 7, 111–120. [Google Scholar] [CrossRef] [PubMed]
  6. Birnbaumer, L. Expansion of signal transduction by G proteins: The second 15 years or so: From 3 to 16 α subunits plus βγ dimers. Biochim. Biochim. Biophys. Acta (BBA) Biomembr. 2007, 1768, 772–793. [Google Scholar] [CrossRef]
  7. Mason, M.G.; Botella, J.R. Completing the heterotrimer: Isolation and characterization of an Arabidopsis thaliana G protein γ-subunit cDNA. Proc. Natl. Acad. Sci. USA 2000, 97, 14784–14788. [Google Scholar] [CrossRef]
  8. Weiss, C.A.; Garnaat, C.W.; Mukai, K.; Hu, Y.; Ma, H. Isolation of cDNAs encoding guanine nucleotide-binding protein beta-subunit homologues from maize (ZGB1) and Arabidopsis (AGB1). Proc. Natl. Acad. Sci. USA 1994, 91, 9554–9558. [Google Scholar] [CrossRef]
  9. Ma, H.; Yanofsky, M.F.; Meyerowitz, E.M. Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 1990, 87, 3821–3825. [Google Scholar] [CrossRef]
  10. Cantos, C.F.; dePamphilis, C.W.; Assmann, S.M. Extra-large G proteins have extra-large effects on agronomic traits and stress tolerance in maize and rice. Trends Plant Sci. 2023, 28, 1033–1044. [Google Scholar] [CrossRef]
  11. Lee, Y.-R.J.; Assmann, S.M. Arabidopsis thaliana ‘extra-large GTP-binding protein’ (AtXLG1): A new class of G-protein. Plant Mol. Biol. 1999, 40, 55–64. [Google Scholar] [CrossRef] [PubMed]
  12. Kankanamge, D.; Tennakoon, M.; Karunarathne, A.; Gautam, N. G protein gamma subunit, a hidden master regulator of GPCR signaling. J. Biol. Chem. 2022, 298, 102618. [Google Scholar] [CrossRef]
  13. Gilman, A.G. G proteins: Transducers of receptor-generated signals. Annu. Rev. Biochem. 1987, 56, 615–649. [Google Scholar] [CrossRef] [PubMed]
  14. Liu, Y.; Wang, X.; Dong, D.; Guo, L.; Dong, X.; Leng, J.; Zhao, B.; Guo, Y.-D.; Zhang, N. Research Advances in Heterotrimeric G-Protein α Subunits and Uncanonical G-Protein Coupled Receptors in Plants. Int. J. Mol. Sci. 2021, 22, 8678. [Google Scholar] [CrossRef]
  15. Jiang, K.; Frick-Cheng, A.; Trusov, Y.; Delgado-Cerezo, M.; Rosenthal, D.M.; Lorek, J.; Panstruga, R.; Booker, F.L.; Botella, J.R.; Molina, A.; et al. Dissecting Arabidopsis Gβ Signal Transduction on the Protein Surface. Plant Physiol. 2012, 159, 975–983. [Google Scholar] [CrossRef] [PubMed]
  16. Trusov, Y.; Chakravorty, D.; Botella, J.R. Diversity of heterotrimeric G-protein γ subunits in plants. BMC Res. Notes 2012, 5, 608. [Google Scholar] [CrossRef]
  17. Subramaniam, G.; Trusov, Y.; Lopez-Encina, C.; Hayashi, S.; Batley, J.; Botella, J.R. Type B Heterotrimeric G Protein γ-Subunit Regulates Auxin and ABA Signaling in Tomato. Plant Physiol. 2015, 170, 1117–1134. [Google Scholar] [CrossRef] [PubMed]
  18. Zhu, H.; Li, G.-J.; Ding, L.; Cui, X.; Berg, H.; Assmann, S.M.; Xia, Y. Arabidopsis Extra Large G-Protein 2 (XLG2) Interacts with the Gβ; Subunit of Heterotrimeric G Protein and Functions in Disease Resistance. Mol. Plant 2009, 2, 513–525. [Google Scholar] [CrossRef]
  19. Jin, Y.-N.; Cui, Z.-h.; Ma, K.; Yao, J.-L.; Ruan, Y.-Y.; Guo, Z.-F. Characterization of ZmCOLD1, novel GPCR-Type G Protein genes involved in cold stress from Zea mays L. and the evolution analysis with those from other species. Physiol. Mol. Biol. Plants 2021, 27, 619–632. [Google Scholar] [CrossRef]
  20. Ma, Y.; Dai, X.; Xu, Y.; Luo, W.; Zheng, X.; Zeng, D.; Pan, Y.; Lin, X.; Liu, H.; Zhang, D.; et al. COLD1 Confers Chilling Tolerance in Rice. Cell 2015, 160, 1209–1221. [Google Scholar] [CrossRef]
  21. Xiong, X.-X.; Liu, Y.; Zhang, L.-L.; Li, X.-J.; Zhao, Y.; Zheng, Y.; Yang, Q.-H.; Yang, Y.; Min, D.-H.; Zhang, X.-H. G-Protein β-Subunit Gene TaGB1-B Enhances Drought and Salt Resistance in Wheat. Int. J. Mol. Sci. 2023, 24, 7337. [Google Scholar] [CrossRef] [PubMed]
  22. Zhang, B.; Zhu, W.; Diao, S.; Wu, X.; Lu, J.; Ding, C.; Su, X. The poplar pangenome provides insights into the evolutionary history of the genus. Commun. Biol. 2019, 2, 215. [Google Scholar] [CrossRef] [PubMed]
  23. Qiu, D.; Bai, S.; Ma, J.; Zhang, L.; Shao, F.; Zhang, K.; Yang, Y.; Sun, T.; Huang, J.; Zhou, Y.; et al. The genome of Populus alba x Populus tremula var. glandulosa clone 84K. DNA Res. 2019, 26, 423–431. [Google Scholar] [CrossRef] [PubMed]
  24. Roy Choudhury, S.; Marlin, M.A.; Pandey, S. The Role of Gβ Protein in Controlling Cell Expansion via Potential Interaction with Lipid Metabolic Pathways. Plant Physiol. 2019, 179, 1159–1175. [Google Scholar] [CrossRef]
  25. Guo, S.; Wang, Y.; Wu, J.; Zhou, X.; Gao, H. Heterotrimeric G-proteins: Multi-dimensional regulation in plant growth, development and abiotic stress responses. Stress Biol. 2025, 5, 3. [Google Scholar] [CrossRef]
  26. Han, A.; Xu, Z.; Cai, Z.; Zheng, Y.; Chen, M.; Wu, L.; Shen, Q. Genome-Wide Identification and Expression Analysis of the G-Protein Gene Family in Barley Under Abiotic Stresses. Plants 2024, 13, 3521. [Google Scholar] [CrossRef]
  27. Gawande, N.D.; Hamiditabar, Z.; Brunetti, S.C.; Gulick, P.J. Characterization of the heterotrimeric G protein gene families in Triticum aestivum and related species. 3 Biotech 2022, 12, 99. [Google Scholar] [CrossRef]
  28. Bisht, N.C.; Jez, J.M.; Pandey, S. An elaborate heterotrimeric G-protein family from soybean expands the diversity of plant G-protein networks. New Phytol. 2011, 190, 35–48. [Google Scholar] [CrossRef]
  29. Harding, S.A.; Hu, H.; Nyamdari, B.; Xue, L.-J.; Naran, R.; Tsai, C.-J. Tubulins, rhythms and cell walls in poplar leaves: It’s all in the timing. Tree Physiol. 2018, 38, 397–408. [Google Scholar] [CrossRef]
  30. Xue, L.-J.; Frost, C.J.; Tsai, C.-J.; Harding, S.A. Drought response transcriptomes are altered in poplar with reduced tonoplast sucrose transporter expression. Sci. Rep. 2016, 6, 33655. [Google Scholar] [CrossRef]
  31. Liu, B.; Zhang, J.; Yang, Z.; Matsui, A.; Seki, M.; Li, S.; Yan, X.; Kohnen, M.V.; Gu, L.; Prasad, K. PtWOX11 acts as master regulator conducting the expression of key transcription factors to induce de novo shoot organogenesis in poplar. Plant Mol. Biol. 2018, 98, 389–406. [Google Scholar] [CrossRef] [PubMed]
  32. Shu, W.; Zhou, H.; Jiang, C.; Zhao, S.; Wang, L.; Li, Q.; Yang, Z.; Groover, A.; Lu, M.Z. The auxin receptor TIR 1 homolog (Pag FBL 1) regulates adventitious rooting through interactions with Aux/IAA 28 in Populus. Plant Biotechnol. J. 2019, 17, 338–349. [Google Scholar] [CrossRef]
  33. Ullah, H.; Chen, J.-G.; Temple, B.; Boyes, D.C.; Alonso, J.M.; Davis, K.R.; Ecker, J.R.; Jones, A.M. The β-Subunit of the Arabidopsis G Protein Negatively Regulates Auxin-Induced Cell Division and Affects Multiple Developmental Processes. Plant Cell 2003, 15, 393–409. [Google Scholar] [CrossRef] [PubMed]
  34. Yang, G.; Wang, S.; Long, L.; Yu, X.; Cai, H.; Chen, P.; Gu, L.; Yang, M. Genome-wide identification and expression analysis of PtJAZ gene family in poplar (Populus trichocarpa). BMC Genom. Data 2023, 24, 55. [Google Scholar] [CrossRef] [PubMed]
  35. Schultheiss, H.; Dechert, C.; Kogel, K.-H.; Hückelhoven, R. Functional analysis of barley RAC/ROP G-protein family members in susceptibility to the powdery mildew fungus. Plant J. 2003, 36, 589–601. [Google Scholar] [CrossRef]
  36. Chen, Y.; Zhang, Y.; Li, S.; Liu, K.; Li, G.; Zhang, D.; Lv, B.; Gu, J.; Zhang, H.; Yang, J.; et al. OsRGA1 optimizes photosynthate allocation for roots to reduce methane emissions and improve yield in paddy ecosystems. Soil Biol. Biochem. 2021, 160, 108344. [Google Scholar] [CrossRef]
  37. Biswas, S.; Islam, M.N.; Sarker, S.; Tuteja, N.; Seraj, Z.I. Overexpression of heterotrimeric G protein beta subunit gene (OsRGB1) confers both heat and salinity stress tolerance in rice. Plant Physiol. Biochem. 2019, 144, 334–344. [Google Scholar] [CrossRef]
  38. Shi, T.; Zhang, X.; Hou, Y.; Jia, C.; Dan, X.; Zhang, Y.; Jiang, Y.; Lai, Q.; Feng, J.; Feng, J.; et al. The super-pangenome of Populus unveils genomic facets for its adaptation and diversification in widespread forest trees. Mol. Plant 2024, 17, 725–746. [Google Scholar] [CrossRef]
  39. Kostiou, V.D.; Theodoropoulou, M.C.; Hamodrakas, S.J. GprotPRED: Annotation of Gα, Gβ and Gγ subunits of G-proteins using profile Hidden Markov Models (pHMMs) and application to proteomes. Biochim. Biophys. Acta (BBA) Proteins Proteom. 2016, 1864, 435–440. [Google Scholar] [CrossRef]
  40. Pandey, S. Plant receptor-like kinase signaling through heterotrimeric G-proteins. J. Exp. Bot. 2020, 71, 1742–1751. [Google Scholar] [CrossRef]
  41. Zhang, W. Roles of heterotrimeric G proteins in guard cell ion channel regulation. Plant Signal. Behav. 2011, 6, 986–990. [Google Scholar] [CrossRef] [PubMed]
  42. Ma, M.; Wang, W.; Fei, Y.; Cheng, H.-Y.; Song, B.; Zhou, Z.; Zhao, Y.; Zhang, X.; Li, L.; Chen, S.; et al. A surface-receptor-coupled G protein regulates plant immunity through nuclear protein kinases. Cell Host Microbe 2022, 30, 1602–1614.e5. [Google Scholar] [CrossRef]
  43. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410. [Google Scholar] [CrossRef]
  44. Finn, R.D.; Clements, J.; Eddy, S.R. HMMER web server: Interactive sequence similarity searching. Nucleic Acids Res. 2011, 39 (Suppl. 2), W29–W37. [Google Scholar] [CrossRef]
  45. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef] [PubMed]
  46. Thompson, J.D.; Gibson, T.J.; Higgins, D.G. Multiple sequence alignment using ClustalW and ClustalX. Curr. Protoc. Bioinform. 2003, 1, 2.3.1–2.3.22. [Google Scholar] [CrossRef]
  47. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef] [PubMed]
  48. Lee, T.-H.; Tang, H.; Wang, X.; Paterson, A.H. PGDD: A database of gene and genome duplication in plants. Nucleic Acids Res. 2012, 41, D1152–D1158. [Google Scholar] [CrossRef]
  49. Wang, Y.; Tang, H.; DeBarry, J.D.; Tan, X.; Li, J.; Wang, X.; Lee, T.-h.; Jin, H.; Marler, B.; Guo, H. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012, 40, e49. [Google Scholar] [CrossRef]
  50. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef]
  51. Kim, D.; Paggi, J.M.; Park, C.; Bennett, C.; Salzberg, S.L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 2019, 37, 907–915. [Google Scholar] [CrossRef] [PubMed]
  52. Li, H.; Handsaker, B.; Wysoker, A.; Fennell, T.; Ruan, J.; Homer, N.; Marth, G.; Abecasis, G.; Durbin, R.; Subgroup, G.P.D.P. The sequence alignment/map format and SAMtools. Bioinformatics 2009, 25, 2078–2079. [Google Scholar] [CrossRef] [PubMed]
  53. Liao, Y.; Smyth, G.K.; Shi, W. featureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 2014, 30, 923–930. [Google Scholar] [CrossRef] [PubMed]
  54. Kolde, R.; Kolde, M.R. Package ‘pheatmap’. R Package 2015, 1, 790. [Google Scholar]
Forests 16 00805 g001
Figure 1. The chromosome distribution of G-protein genes in the poplar genome. (A) The chromosome distribution of G-protein genes in subgenome A (P. alba); (B) the chromosome distribution of G-protein genes in subgenome G (P. tremula var. glandulosa). Each colored vertical bar represents a chromosome, with chromosome length (Mb) shown on the left. Gene names are labeled next to their corresponding chromosomal positions. Ten chromosomes (chr01, chr03, chr04, chr05, chr06, chr07, chr09, chr12, chr15 and chr18) harbor G-protein genes in both subgenome A and G.
Figure 1. The chromosome distribution of G-protein genes in the poplar genome. (A) The chromosome distribution of G-protein genes in subgenome A (P. alba); (B) the chromosome distribution of G-protein genes in subgenome G (P. tremula var. glandulosa). Each colored vertical bar represents a chromosome, with chromosome length (Mb) shown on the left. Gene names are labeled next to their corresponding chromosomal positions. Ten chromosomes (chr01, chr03, chr04, chr05, chr06, chr07, chr09, chr12, chr15 and chr18) harbor G-protein genes in both subgenome A and G.
Forests 16 00805 g001
Forests 16 00805 g002
Figure 2. Phylogenetic tree of G-proteins in Arabidopsis, rice and poplar. Protein sequences from 27 poplar G-proteins, 8 Arabidopsis G-proteins and 11 G-proteins in rice were aligned using ClustalW. A Neighbor-Joining (NJ) phylogenetic tree of the G-protein gene family was constructed using MEGA with a bootstrap value of 1000. The red branches represent the Gα subgroup; the blue branches represent Gβ subgroup; the yellow branches represent Gγ subgroup.
Figure 2. Phylogenetic tree of G-proteins in Arabidopsis, rice and poplar. Protein sequences from 27 poplar G-proteins, 8 Arabidopsis G-proteins and 11 G-proteins in rice were aligned using ClustalW. A Neighbor-Joining (NJ) phylogenetic tree of the G-protein gene family was constructed using MEGA with a bootstrap value of 1000. The red branches represent the Gα subgroup; the blue branches represent Gβ subgroup; the yellow branches represent Gγ subgroup.
Forests 16 00805 g002
Forests 16 00805 g003
Figure 3. Gene structure, conserved domain and motif analysis of G-protein in poplar. (A) A Neighbor-Joining (NJ) phylogenetic tree constructed using the full-length of protein sequences of G-protein gene family in poplar. The bootstraps value was set to 1000. The blue branches represent Gα proteins; the green branches represent Gβ proteins; the red branches represent Gγ proteins. (B) Conserved motif analysis of G-protein in poplar. A total of 20 motifs were identified using MEME website tool and named as Motif 1-Motif 20. The scale bar at the bottom represents the amino acid number. (C) Gene structure analysis of Gα protein genes in poplar, revealing the organization of UTR (yellow), CDSs (green) and introns (grey). The scale bar at the bottom represents the length of genomic sequences. (D) Conserved domain analysis of G-protein in poplar using CDD website tools. A total of five types of conserved domains were detected in G-protein, including G-alpha, FYVE_linke_SF superfamily, G-gamma, G-gamma superfamily, PTZ00169 superfamily and WD40. The scale bar at the bottom represents the amino acid number.
Figure 3. Gene structure, conserved domain and motif analysis of G-protein in poplar. (A) A Neighbor-Joining (NJ) phylogenetic tree constructed using the full-length of protein sequences of G-protein gene family in poplar. The bootstraps value was set to 1000. The blue branches represent Gα proteins; the green branches represent Gβ proteins; the red branches represent Gγ proteins. (B) Conserved motif analysis of G-protein in poplar. A total of 20 motifs were identified using MEME website tool and named as Motif 1-Motif 20. The scale bar at the bottom represents the amino acid number. (C) Gene structure analysis of Gα protein genes in poplar, revealing the organization of UTR (yellow), CDSs (green) and introns (grey). The scale bar at the bottom represents the length of genomic sequences. (D) Conserved domain analysis of G-protein in poplar using CDD website tools. A total of five types of conserved domains were detected in G-protein, including G-alpha, FYVE_linke_SF superfamily, G-gamma, G-gamma superfamily, PTZ00169 superfamily and WD40. The scale bar at the bottom represents the amino acid number.
Forests 16 00805 g003
Forests 16 00805 g004
Figure 4. The cis-acting elements on the promoters of G-protein genes in poplar. (A) The distribution of cis-acting elements in the putative promoters (upstream 2k) of G-protein genes in poplar. The phylogenetic tree was constructed using MEGA with the NJ method (bootstraps value = 1000). (B) The number of cis-acting elements in the promoters of genes. A total of 18 key cis-acting elements were detected and analyzed in this study, including auxin-responsive element, cis-acting element involved in defense and stress responsiveness, cis-acting element involved in low-temperature responsiveness, cis-acting element involved in phytochrome down-regulation expression, cis-acting element involved in salicylic acid responsiveness, cis-acting element involved in the abscisic acid responsiveness, cis-acting regulatory element essential for the anaerobic induction, cis-acting regulatory element involved in circadian control, cis-acting regulatory element involved in seed-specific regulation, cis-acting regulatory element involved in the MeJA-responsiveness, cis-acting regulatory element involved in zein metabolism regulation, cis-acting regulatory element related to meristem expression, gibberellin-responsive element, light responsiveness, the MYB binding site involved in drought-inducibility, the MYB binding site involved in flavonoid biosynthetic genes regulation, the MYB binding site involved in light responsiveness and the MYBHv1 binding site.
Figure 4. The cis-acting elements on the promoters of G-protein genes in poplar. (A) The distribution of cis-acting elements in the putative promoters (upstream 2k) of G-protein genes in poplar. The phylogenetic tree was constructed using MEGA with the NJ method (bootstraps value = 1000). (B) The number of cis-acting elements in the promoters of genes. A total of 18 key cis-acting elements were detected and analyzed in this study, including auxin-responsive element, cis-acting element involved in defense and stress responsiveness, cis-acting element involved in low-temperature responsiveness, cis-acting element involved in phytochrome down-regulation expression, cis-acting element involved in salicylic acid responsiveness, cis-acting element involved in the abscisic acid responsiveness, cis-acting regulatory element essential for the anaerobic induction, cis-acting regulatory element involved in circadian control, cis-acting regulatory element involved in seed-specific regulation, cis-acting regulatory element involved in the MeJA-responsiveness, cis-acting regulatory element involved in zein metabolism regulation, cis-acting regulatory element related to meristem expression, gibberellin-responsive element, light responsiveness, the MYB binding site involved in drought-inducibility, the MYB binding site involved in flavonoid biosynthetic genes regulation, the MYB binding site involved in light responsiveness and the MYBHv1 binding site.
Forests 16 00805 g004
Forests 16 00805 g005
Figure 5. The evolution analysis of the G-protein gene family. (A) The number and proportion of genes produced by five types of duplication events in subgenome A and G, including WGD or segmental duplication, singleton duplication, dispersed duplication, proximal duplication and tandem duplication. (B) The heatmap represents the duplication events of G-protein genes in subgenome A and G. (C) Distribution and collinearity of the G-protein gene family in subgenome A and G. From the outer edge inward: (I) circles represent the chromosome; (II) circles represent the gene density (window size = 500 k); and (III) circles represent the TE density (window size = 500 k). The circle pictures were plotted by circos software. The red line shows the collinearity gene pair of G-protein genes in subgenome A; the blue line shows the collinearity gene pair of G-protein genes in subgenome G; the grey line shows the collinearity gene pair of in poplar genome.
Figure 5. The evolution analysis of the G-protein gene family. (A) The number and proportion of genes produced by five types of duplication events in subgenome A and G, including WGD or segmental duplication, singleton duplication, dispersed duplication, proximal duplication and tandem duplication. (B) The heatmap represents the duplication events of G-protein genes in subgenome A and G. (C) Distribution and collinearity of the G-protein gene family in subgenome A and G. From the outer edge inward: (I) circles represent the chromosome; (II) circles represent the gene density (window size = 500 k); and (III) circles represent the TE density (window size = 500 k). The circle pictures were plotted by circos software. The red line shows the collinearity gene pair of G-protein genes in subgenome A; the blue line shows the collinearity gene pair of G-protein genes in subgenome G; the grey line shows the collinearity gene pair of in poplar genome.
Forests 16 00805 g005
Forests 16 00805 g006
Figure 6. The heatmap showing the expression patterns of 27 G-protein genes in different tissues of poplar. The heatmap illustrates the expression patterns of 27 G-protein genes across different tissues in ‘P. tremula × P. alba’ (A) and ‘P. alba × P. tremula var. glandulosa’ (B). Red color indicates high expression genes, while blue indicates low expression genes. The color scale bar represents Fragments Per Kilobase of exon per Million (FPKM) values normalized by ‘scale’ function in R language. The heatmaps were plotted using the pheatmap package in R.
Figure 6. The heatmap showing the expression patterns of 27 G-protein genes in different tissues of poplar. The heatmap illustrates the expression patterns of 27 G-protein genes across different tissues in ‘P. tremula × P. alba’ (A) and ‘P. alba × P. tremula var. glandulosa’ (B). Red color indicates high expression genes, while blue indicates low expression genes. The color scale bar represents Fragments Per Kilobase of exon per Million (FPKM) values normalized by ‘scale’ function in R language. The heatmaps were plotted using the pheatmap package in R.
Forests 16 00805 g006
Forests 16 00805 g007
Figure 7. The heatmap showing the expression patterns of 27 G-protein genes under pathogen and abiotic treatments. (A) Expression profiles of G-protein genes under pathogen (A. alternata) treatment. (B) Expression profiles of G-protein genes under four abiotic treatments, including drought, salt, high temperature and low temperature. The color scale bar represents Fragments Per Kilobase of exon per Million (FPKM) values normalized by ‘scale’ function in R language. The heatmaps were plotted using the pheatmap package in R.
Figure 7. The heatmap showing the expression patterns of 27 G-protein genes under pathogen and abiotic treatments. (A) Expression profiles of G-protein genes under pathogen (A. alternata) treatment. (B) Expression profiles of G-protein genes under four abiotic treatments, including drought, salt, high temperature and low temperature. The color scale bar represents Fragments Per Kilobase of exon per Million (FPKM) values normalized by ‘scale’ function in R language. The heatmaps were plotted using the pheatmap package in R.
Forests 16 00805 g007
Table 1. Basic information of the G-protein gene family in poplar.
Table 1. Basic information of the G-protein gene family in poplar.
Gene NameGene IDChromosomeStartEndStrandG Protein Type (Gα, Gβ, Gγ)Number of Amino AcidMolecular WeightTheoretical pIInstability IndexAliphatic IndexGrand Average of HydropathicityPrediction of Subcellular Localization
PagGα1Pop_A01G004180.T1chrA0137,893,88537,899,565+ Gα 86198,2305.6846.2974.24−0.557nuclear
PagGα2Pop_A03G050281.T1chrA039,155,0399,164,024− Gα 85997,8805.6848.0275.32−0.572nuclear
PagGα3Pop_A05G072994.T1chrA0511,557,52411,562,795− Gα 919102,2615.0943.7978.24−0.426nuclear
PagGα4Pop_A06G053386.T1chrA064,522,6324,527,073− Gα 38444,5936.3943.9483.8−0.54cytoplasmic
PagGα5Pop_A07G022806.T1chrA0711,068,51711,074,776− Gα 924103,0035.1142.2977.91−0.434nuclear
PagGα6Pop_A09G026564.T1chrA093,705,3113,712,625+ Gα 918103,1546.0746.0479.81−0.436nuclear
PagGα7Pop_A12G073756.T1chrA129,498,3439,506,833− Gα 83895,9585.2452.4470.87−0.67nuclear
PagGα8Pop_A15G064380.T1chrA1510,744,97210,749,965+ Gα 84596,3895.2353.6770.3−0.651nuclear
PagGα9Pop_A18G012744.T1chrA189,177,7689,183,224+ Gα 39245,5995.7938.4387.27−0.493chloroplast
PagGα10Pop_G01G089285.T1chrG0138,117,47538,123,916+ Gα 86198,2495.6846.7373.9−0.565nuclear
PagGα11Pop_G03G010649.T1chrG0310,432,29310,440,689− Gα 85897,6645.7347.6274.85−0.561nuclear
PagGα12Pop_G05G017901.T1chrG0511,563,25211,569,032− Gα 918102,1925.1443.4578.42−0.423nuclear
PagGα13Pop_G06G003473.T1chrG064,369,4364,375,962− Gα 39245,4946.3943.0585.08−0.513mitochondrial
PagGα14Pop_G07G062643.T1chrG0711,367,11911,372,298− Gα 926103,0785.1342.6578.59−0.423nuclear
PagGα15Pop_G09G077213.T1chrG093,715,6093,722,355+ Gα 918103,0785.9946.8479.72−0.445nuclear
PagGα16Pop_G12G050751.T1chrG129,593,0919,601,147− Gα 83895,8765.2651.7670.75−0.666nuclear
PagGα17Pop_G15G074300.T1chrG1511,083,77211,088,980+ Gα 84596,5775.3255.5170.07−0.667nuclear
PagGα18Pop_G18G011084.T1chrG1810,021,60810,026,716+ Gα 42148,8105.9142.4284.96−0.553cytoplasmic
PagGβ1Pop_A04G028317.T1chrA045,919,1465,923,477+ Gβ 41044,9596.4536.1978.66−0.25nuclear
PagGβ2Pop_A09G026627.T1chrA093,355,6133,359,495+ Gβ 39742,9656.2631.3386.85−0.094nuclear
PagGβ3Pop_G04G028074.T1chrG046,276,1026,279,679+ Gβ 28830,9005.8233.3773.09−0.221nuclear
PagGβ4Pop_G09G077148.T1chrG093,370,7273,375,071+ Gβ 37740,7956.6927.3182.18−0.144nuclear
PagGγ1Pop_A15G014669.T1chrA15367,628370,487− Gγ 11012,4094.9141.6770.18−0.677chloroplast
PagGγ2Pop_A18G083136.T1chrA186,983,2886,984,925− Gγ 11112,8159.3780.2772.88−0.452nuclear
PagGγ3Pop_G06G051470.T1chrG0612,624,04112,626,469− Gγ 25428,0439.3958.9191.810.07nuclear
PagGγ4Pop_G15G034021.T1chrG15265,063267,901− Gγ 11012,4094.9141.5970.18−0.701chloroplast
PagGγ5Pop_G18G085849.T1chrG187,853,2047,859,568+ Gγ 38142,0009.7554.1588.64−0.076cytoplasmic
Table 2. The collinearity gene pair of G-protein genes in subgenome A and subgenome G.
Table 2. The collinearity gene pair of G-protein genes in subgenome A and subgenome G.
SubgenomeChromosomeStartEndGene1ChromosomeStartEndGene2
P. alba (subgenome A)chrA0511,557,524.0011,562,795.00PagGα3chrA0711,068,517.0011,074,776.00PagGα5
chrA0511,557,524.0011,562,795.00PagGα3chrA093,705,311.003,712,625.00PagGα6
chrA0711,068,517.0011,074,776.00PagGα5chrA093,705,311.003,712,625.00PagGα6
chrA129,498,343.009,506,833.00PagGα7chrA1510,744,972.0010,749,965.00PagGα8
P. tremula var. glandulosa (subgenome G)chrG0138,117,475.0038,123,916.00PagGα10chrG0310,432,293.0010,440,689.00PagGα11
chrG046,276,102.006,279,679.00PagGβ3chrG093,370,727.003,375,071.00PagGβ4
chrG0511,563,252.0011,569,032.00PagGα12chrG0711,367,119.0011,372,298.00PagGα14
chrG0511,563,252.0011,569,032.00PagGα12chrG093,715,609.003,722,355.00PagGα15
chrG064,369,436.004,375,962.00PagGα13chrG1810,021,608.0010,026,716.00PagGα18
chrG0612,624,041.0012,626,469.00PagGγ3chrG187,853,204.007,859,568.00PagGγ5
chrG0711,367,119.0011,372,298.00PagGα14chrG093,715,609.003,722,355.00PagGα15
chrG129,593,091.009,601,147.00PagGα16chrG1511,083,772.0011,088,980.00PagGα17
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Song, B.; Liu, Q.; Zeng, Z.; Gu, Y.; Ye, W.; Fu, F.; Ming, M. Genome-Wide Identification, Evolution and Expression Analysis of the G-Protein Gene Family in Poplar (Populus alba × Populus glandulosa). Forests 2025, 16, 805. https://doi.org/10.3390/f16050805

AMA Style

Song B, Liu Q, Zeng Z, Gu Y, Ye W, Fu F, Ming M. Genome-Wide Identification, Evolution and Expression Analysis of the G-Protein Gene Family in Poplar (Populus alba × Populus glandulosa). Forests. 2025; 16(5):805. https://doi.org/10.3390/f16050805

Chicago/Turabian Style

Song, Bobo, Qian Liu, Zitong Zeng, Yiyang Gu, Wenxin Ye, Fangfang Fu, and Meiling Ming. 2025. "Genome-Wide Identification, Evolution and Expression Analysis of the G-Protein Gene Family in Poplar (Populus alba × Populus glandulosa)" Forests 16, no. 5: 805. https://doi.org/10.3390/f16050805

APA Style

Song, B., Liu, Q., Zeng, Z., Gu, Y., Ye, W., Fu, F., & Ming, M. (2025). Genome-Wide Identification, Evolution and Expression Analysis of the G-Protein Gene Family in Poplar (Populus alba × Populus glandulosa). Forests, 16(5), 805. https://doi.org/10.3390/f16050805

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

Article Metrics

Back to TopTop