1. Introduction
Peas (Pisum sativum L.) are a globally significant legume that play a vital role in human nutrition and serve as a protein-rich feed for livestock. Despite their importance, genomic research focusing on peas remains limited due to the complexity of their genetic makeup. However, recent breakthroughs in genomic technologies, such as the creation of genetic resource databases, the advent of high-throughput genotyping techniques, and the assembly of reference genomes, have opened new avenues for pea genomics [1]. These advances have not only shed light on the domestication and breeding history of peas but have also assisted in the discovery of genes linked to key traits [2]. For instance, the sequencing of the pea genome has marked a significant leap forward in our theoretical understanding with practical applications in plant breeding [3]. Historically, the European Union has been a major producer of peas, accounting for a substantial portion of the global production. With the anticipated rise in demand for vegetable protein by 2035, the significance of the global pea market is expected to expand further [4]. Yet, the exploration of pea genomics and gene functionality, particularly in the context of seed nutrition and abiotic stress responses, continues to present scientific challenges.
The enzyme trehalose-6-phosphate synthase (TPS) plays an integral role in pea seed development [5]. It catalyzes the synthesis of trehalose-6-phosphate synthase (TPS), a critical signaling and regulatory molecule implicated in plant growth, development, and stress responses [6]. Furthermore, it regulates sucrose levels, impacts its synthesis, and aligns the growth of sink organs with the availability of sucrose [7]. There is a pressing need to unravel the significance of TPS in seed development in order to enhance our comprehension of peas as a crop and to develop more effective breeding and seed production strategies.
In the realm of abiotic stress responses, TPS genes have emerged as pivotal in various plant species. For instance, in sesame, quinoa, soybean, rapeseed, and pepper, TPS gene expression is significantly modulated under different stress conditions, such as drought, salt stress, and osmotic stress, highlighting their contribution to stress adaptations [8,9,10,11,12]. The SiTPS10 gene in sesame, for example, is upregulated under multiple stress conditions, suggesting its key role in stress tolerance [8]. In rapeseed, ArTPS genes are implicated in the response to NaCl stress, thereby influencing the metabolism of polysaccharides and glycosides and the accumulation of glycyrrhizin [11]. Despite similar findings in other plants, the specific functions of the TPS genes and their involvement in stress responses in peas remain elusive. Given the agronomic importance of peas and the potential of TPS genes to enhance their resilience to environmental stressors, a detailed examination of these genes in peas is both timely and necessary.
This study leveraged cutting-edge genomic data and bioinformatic tools to identify and characterize 20 PsTPS genes in wheat. Phylogenetic analysis, gene structure examination, conserved domain analysis, and interaction network predictions were performed to explore the evolutionary and structural attributes of PsTPSs. Additionally, the expression profiles of PsTPSs were systematically investigated across 20 RNA-seq samples, with a select group of hormone-responsive candidates further validated via transcriptome sequencing analysis. This study lays down a theoretical reference for future functional studies on PsTPSs and elucidates their roles in hormone responses and plant growth regulation.
2. Materials and Methods
2.1. Identification and Sequence Analysis of TPS Genes from Pisum sativum
We procured the complete genomic sequence of the peas from the National Center for Biotechnology Information (NCBI,
To substantiate the authenticity of these candidate sequences, we engaged the NCBI Conserved Domain Database (CDD) and the SMART tool, both employing an E-value threshold of 10−5 and employing filters that targeted the TPS domain sequence. The TPS genes that passed this validation were designated new names which were reflective of their chromosomal coordinates within the pea genome.
For the prediction of the subcellular destinations of TPS proteins, we deployed the Cell-PLoc software (v2.0). Concurrently, the ExPASy ProtParam tool was harnessed to forecast a spectrum of physicochemical properties of the proteins, encompassing molecular weight (MW), isoelectric point (pI), instability index, and grand average of hydropathicity (GRAVY), providing a comprehensive profile of the protein characteristics of each TPS.
2.2. Phylogenetic Relationship, Gene Structure and Conserved Motifs Analysis
To elucidate the phylogenetic ties among the TPS genes of peas, a series of multi-sequence alignments were executed on the recognized TPS proteins from the peas, alongside those from Manihot esculenta, Populus trichocarpa, and Arabidopsis. The ClustalW software (Version 2.1) was the instrument of choice for these alignments. Subsequently, the sequences were subjected to IQ-TREE 2, which applied the maximum likelihood (ML) method to construct an evolutionary tree, reinforced with a bootstrap value of 1000 for statistical support.
The precise chromosomal locales and the exon–intron architectures of the TPS genes under scrutiny were sourced from the annotated genome files, obtained from the Ensembl Plants database (
In addition to the structural and localization analyses, the MEME suite was engaged to forecast the conserved motifs within the sequence of each TPS protein. The search parameters were set to identify a maximum of 15 motifs, with all other settings adhering to the default configurations of the MEME tool (
2.3. Chromosomal Distribution, Syntenic Analysis, and Predicting the Protein–Protein Interaction Network of the PsTPSs
The downstream analysis function in MCScanX (Version 2.0) was used to calculate the Ka and Ks of the segmental and tandem duplicate gene pairs. The Ks values were used to calculate the dates of duplication events (T) according to the following equation: T = Ks/2λ, with λ = 1.5 × 10−8 s for dicots [15]. The Ka/Ks value was further employed to identify the selection mode of the PsTPSs. STRING (
The positional and structural details of genes mapped onto the chromosomes of pea were sourced from the GFF3 file, which was obtained from the peas genome database (
2.4. Material and Treatments
The cultivation of the TM-1 seeds of the ‘Zhewan No.1’ (ZW1) pea. variety commenced in a fertile matrix of soil within a controlled-environment greenhouse. The greenhouse was programmed to offer a 16 h photoperiod at a constant temperature of 27 °C, succeeded by an 8 h period of darkness at 22 °C, with relative humidity levels sustained between 60% and 80%. Once the TM-1 pea seedlings had developed to a stage marked by the emergence of three leaves, and exhibiting signs of vigorous health and consistent growth, they were subjected to experimental conditions aimed at eliciting responses to drought and saline stress.
To elicit salt stress, the root systems of the seedlings were irrigated with a solution containing 300 mM of sodium chloride (NaCl). Conversely, to simulate drought conditions, the roots were treated with a 20% solution of polyethylene glycol 6000 (PEG6000). Three-leaf-stage pea (Pisum sativum L.) seedlings were cultivated in a 70% vermiculite and 30% peat moss mixture. Group 1 received a 300 mM NaCl solution for 24 h to induce salinity stress. Group 2 was treated with a 20% PEG6000 and 300 mM NaCl solution for 24 h to simulate drought stress. A control group was watered normally. Leaf samples were collected at 0 and 24 h post treatment for further analysis. Each leaf sample was promptly submerged in liquid nitrogen for flash-freezing and then securely preserved at −80 °C, ensuring the integrity of the RNA for subsequent RNA extraction and experimental analysis.
2.5. Transcriptome Sequencing
After the treatment at both the 3 h and 24 h marks, the pea seedling leaves from both the control group without treatment and those treated with 300 mmol NaCl solution for the respective durations, along with leaves treated with a 20% concentration solution of PEG6000, were rapidly frozen in liquid nitrogen and sent to Tianjin Jizhi Gene Technology Co., Ltd. (Tianjin, China) for advanced analysis. To ensure experimental reliability, three biological replicates were performed. The total RNA from these leaf samples was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The RNA integrity was evaluated by agarose gel electrophoresis, where the total RNA exhibited clear 28S and 18S rRNA bands with the 28S band approximately twice as intense as the 18S band. Subsequently, the samples were sequenced using the Illumina Novaseq 6000 platform. Library construction was performed in accordance with the protocols provided in the Illumina TruSeq RNA Sample Prep Kit. The RNA sequencing libraries were prepared through a series of steps including total RNA extraction, mRNA enrichment and fragmentation, cDNA synthesis, double-stranded cDNA end-repair, adapter ligation, and PCR enrichment.
The raw sequencing reads were subjected to a rigorous quality control and were cleaned to ensure high quality. These refined reads were then aligned to the reference genome, accessible through the specified link, to assemble transcripts and measure gene expression levels. The quality of the RNA-seq alignments was carefully reviewed. The RNA-seq data were analyzed using Weighted Gene Co-expression Network Analysis (WGCNA) to assess and standardize the transcriptomic data quality. The subsequent transcriptome analysis was conducted on a specialized cloud-based platform developed by Tianjin Jizhi Gene Technology Co., Ltd., ensuring a thorough and detailed examination of the data.
3. Results
3.1. Whole-Genome Characterization of TPS Protein in Pisum sativum
In the current study, the hidden Markov model (HMM) specific to plant trehalose-6-phosphate synthase (TPS) proteins was initially harnessed and employed to conduct a genome-wide search of the protein sequences of peas. This was followed by a validation process to verify the authenticity of the identified candidate TPS proteins. A total of 20 TPS proteins were identified, each featuring a conserved TPS domain, which was classified as part of the TPS family in peas. These proteins were sequentially termed PsTPS1 through to PsTPS20, in line with their chromosomal positions (Figure 1, Table S1).
Then, the fundamental characteristics of the PsTPS genes were examined, encompassing parameters such as protein length in amino acids (aa), molecular weight (MW), isoelectric point (pI), instability index, and grand average of hydropathy (GRAVY). The protein lengths varied from a minimum of 194 aa for PsTPS3 to a maximum of 965 aa for PsTPS1, with an overall average length of 541.2 aa. Correspondingly, the molecular weight of the proteins spanned from 20.89 kD for PsTPS3 to 108.366 kD for PsTPS1, averaging 60.97 kD. Notably, PsTPS3 was an outlier with a significantly shorter protein length and lower molecular weight compared to the other PsTPS genes. The isoelectric points of the PsTPS genes extended from 5.65 to 9.64, the instability indices ranged from 14.69 to 51.34, and the GRAVY values, an indicator of protein hydrophobicity, fluctuated between −0.503 and −0.105.
3.2. Phylogenetic Relationships, Gene Structure, Conserved Motif and Cis-Regulatory Elements Analysis of PsTPSs
To gain insights into the evolutionary connections among the TPS gene family members in the peas, represented by 20 PsTPS genes, and their counterparts in Arabidopsis, represented by 20 AtTPS genes, and Glycine max, represented by 37 GmTPS genes, a maximum likelihood (ML) phylogenetic tree was developed to display the phylogenetic relationships among these species, as illustrated in (Figure 2). The 77 TPS genes included in the phylogenetic analysis were categorized into four distinct subfamilies, following the taxonomic classifications and topological structures established in prior research.
Interestingly, the distribution of the 20 PsTPS genes across these subfamilies was non-uniform, as detailed in the Supplementary Material (Table S2). Among the subfamilies, subfamily I was the most populated, with seven PsTPS genes, whereas Subfamily III contained the fewest number of genes, with only three PsTPS genes. This differential distribution within the phylogenetic tree demonstrated the diversity and evolutionary complexity of the TPS gene family across the examined species. The phylogenetic analysis not only provided a framework for understanding the relationships among these genes but also laid the groundwork for future studies investigating the functional significance of these subfamily-specific distributions.
Furthermore, a detailed examination of the gene structures and the arrangement of conserved motifs among the 20 PsTPS proteins was carried out to correlate our findings with their phylogenetic relationships, as depicted in Figure 3a–d. The MEME online tool was utilized to search for conserved motifs within the PsTPS proteins, yielding 10 distinct motifs (motifs 1–10; Figure 3b). This analysis exposed variations in the composition of motifs, with the overall motif distribution closely mirroring the categorization into different subfamilies. Specifically, motifs 5, 9, and 10 were predominantly associated with Subfamily I, whereas motifs 1, 2, and 3 were characteristic of Subfamily IV, and Subfamilies II and III displayed a more diverse array of motifs.
Concurrently, the gene domains of the PsTPS proteins were scrutinized, and the alignment of the conserved TPS domains with the genetic architecture of the peas graphically represented (Figure 3c). Our findings indicated the presence of five distinct types of TPS domains, with most genes anticipated to encompass at least one of two key domains: the “Glycosyltransferase family 20” domain and the “TREHALOSE-6-PHOSPHATE SYNTHASE” domain.
Further analysis of the exon count within the 20 PsTPS genes, based on the GFF annotation file of peas, revealed a range from 2 to 17 exons (Figure 3d). A significant number of PsTPS genes possessed over 10 coding sequences (CDSs). Genes from related subfamilies demonstrated a tendency towards analogous exon/intron configurations, implying a strong correlation between the phylogenetic connections and the structural composition of the genes within the family.
This comprehensive characterization of the PsTPS gene family provides a deeper understanding of their molecular composition and evolutionary interrelations, setting a solid foundation for subsequent functional studies and potential applications in the genetic enhancement of the peas.
Cis-regulatory elements (CREs), comprising non-coding DNA sequences in the gene promoter region, play a decisive role in gene expression and participate in the modulation of various biological processes. In the current study, the 2000 base pair (bp) promoter sequences upstream of the identified PsTPS genes were analyzed to forecast CREs, utilizing the PlantCARE database. A total of 215 CRE sites were extracted from the predictive outcomes and are presented in Figure 4a. Notably, PsTPS17 exhibited the highest count of CREs, totaling 18 distinct elements. A comparison across all subfamilies revealed that elements associated with growth and development were the most prevalent, closely followed by those linked to hormone responsiveness, as depicted in Figure 4b.
3.3. Evolutionary Analyses of the PsTPSs Within and Between Species
To assess the genomic basis of the PsTPS gene family expansion in peas, syntenic relationships among the PsTPS genes were examined. A total of 3 out of the 20 PsTPS genes exhibited syntenic ties, with 2 pairs identified as segmental duplications within the pea genome, as detailed in Table S3. The ratio of non-synonymous to synonymous substitution rates, denoted as Ka/Ks, reflects the selective pressure in gene duplication events. The Ka/Ks ratios for the 2 segmental duplication gene pairs were below 1, signifying that these gene pairs have likely experienced strong negative selection throughout their evolutionary history. In order to enhance our comprehension of genetic divergence, gene duplication, and evolutionary patterns among the TPS gene families of P. sativum, A. thaliana, and G. max, syntenic relationships were further explored to identify orthologous TPS genes among these species using the MCScanX tool. A total of 20 and 49 pairs of orthologous TPS genes were identified across three comparative analyses (P. sativum versus A. thaliana, P. sativum versus G. max), as illustrated in Figure 5. Notably, some genes displayed numerous homologous relationships across different species. Importantly, syntenic genes were predominantly located on chromosomes Ps2, Ps4 and Ps5 to Ps7.
3.4. Interaction Networks Analysis of PsTPSs
To investigate the biological roles and the intricate regulatory networks associated with PsTPSs, potential protein–protein interactions (PPIs) among proteins were predicted using an orthology-based approach. The analysis revealed that 11 PsTPSs shared orthologous relationships with Arabidopsis, suggesting a conserved evolutionary link. Intriguingly, within the PsTPS gene family, interactions were detected among PsTPS15, PsTPS19, PsTPS10, PsTPS5, and PsTPS14, as illustrated in Figure 6. Additionally, PsTPS17 interacted with TPS14A and CDKA-1. Lastly, each of these interacting proteins participated in the regulatory machinery governing plant cell growth, thereby implicating PsTPSs in these processes as well.
3.5. Tissue-Specific Expression Patterns of PsTPS
To elucidate the expression profiles of the PsTPS gene family, an expression heatmap was constructed for the eight PsTPS genes (Figure 7), uncovering distinct tissue-specific expression patterns across various plant tissues, including primary and lateral roots, root nodules, young and mature stems, tendrils, young and mature leaflets along with their stipules, sepals, petals, pods, and seeds, representing the diverse tissue distribution in all 20 PsTPS genes. Moreover, the expression levels of PsTPS15 and PsTPS18 were elevated in primary and lateral roots, root nodules, and young stems, indicating their involvement in root growth initiation and nodule formation, which are essential for nutrient uptake and symbiotic relationships with nitrogen-fixing bacteria. Conversely, the expression of PsTPS13 and PsTPS19 was downregulated in seeds, suggesting tissue-specific functions that warrant further exploration.
3.6. Transcriptional Responses of PsTPS Genes to Drought and Salt Stress in Pisum sativum
To understand the response of the TPS gene family to drought conditions, pea seedlings were exposed to a 20% concentration solution of PEG6000. Samples of leaf tissue were collected at 3 h and 24 h intervals for RNA extraction and transcriptome analysis. After treatment with a 20% concentration PEG6000 solution, a substantial shift was detected in the transcriptomic response of 14 PsTPS genes, with PsTPS4 and PsTPS6 exhibiting pronounced alterations, as illustrated in Figure 8. This expression pattern of an initial increase followed by a decrease reflected an immediate response to dehydration and subsequent acclimation. Early functional assessments suggested that these genes may potentially play key roles in signaling and metabolic pathways critical for drought adaptation.
On the other hand, the expression levels of genes such as PsTPS15 and PsTPS19 progressively decreased, highlighting their importance under prolonged stress conditions. This sustained decrease in expression could be pivotal for sustaining cell hydration and promoting the antioxidant defense system.
Furthermore, salinity stress was explored by exposing seedlings to increasing 300 mmol concentration of NaCl solution concentrations. The analysis demonstrated significant alterations in the expression patterns of several PsTPS genes, with PsTPS4, PsTPS6, and PsTPS19 being particularly impacted, as depicted in Figure 8. These changes are hypothesized to be intimately linked to the plant’s strategies for acclimating to saline conditions, potentially involving an osmotic adjustment and ion homeostasis.
4. Discussion
Trehalose-6-phosphate synthase (TPS) genes are integral components of plant biochemistry, serving as critical catalysts for the synthesis of trehalose, a key component in the defense mechanisms of a plant against environmental stress [8]. Trehalose, characterized by its non-reducing disaccharide structure, is recognized for its protective role against a variety of stressors in living organisms [8]. In Arabidopsis, 11 TPS genes with TPS and TPP-like domains were identified, including the active AtTPS1, revealing the role of the TPS family in plant physiology and abiotic stress responses [16]. Thirty-one ScTPS genes were identified in sugar cane, exhibiting differential expression in response to simulated drought, salinity, and ABA stress [17]. References are placed in the appendix. Within the plant kingdom, TPS genes are not only pivotal for the synthesis of trehalose but also play a responsive role under stress conditions, such as those induced by high salinity [8]. They also hold significant implications in plant metabolic pathways, including those related to glycometabolism and the management of polysaccharides and glycosides [8]. Of note, these genes have been discovered and described across a spectrum of plant species, spanning from sesame to rapeseed, quinoa, and soybean [5,9,10], and are distributed across various chromosomes with potentially diverse cellular locations. Through phylogenetic evaluation, gene structural analysis, and the identification of conserved motifs, TPS genes have been classified into different categories. Expression studies have unveiled patterns of tissue specificity and reactivity to stress, with certain genes, such as SiTPS10 in sesame and BnTPSs in rapeseed, showing potential in optimizing plant resilience against a range of abiotic stresses [8]. In summary, TPS is a fundamental factor in the regulation of plant growth, development, and adaptation to environmental stressors.
Our research offers an in-depth examination of the diversity in structure and function of the Trehalose-6-phosphate synthase (TPS) genes within peas. It emphasizes the preservation of characteristics within TPS subfamilies and the divergence observed across different species while also elucidating gene duplication and the broader evolutionary context. Through our investigation, 20 pea genes responsible for encoding Trehalose-6-phosphate synthase were identified and categorized into four subfamilies using phylogenetic analysis. The findings exposed both similarities and differences in TPS genes compared with other plant species, indicating a diversification in structure and function across the examined plant species.
The analysis of motifs and gene structures revealed that TPS genes with close phylogenetic relationships were more likely to have analogous motif compositions and exon–intron arrangements, a pattern also noted in species such as quinoa and Brassica napus L. A significant variation in the count of exons and introns was observed in PsTPSs, with numbers ranging from 0 to 17, echoing the variability observed in species such as Brassica rapa L. Meanwhile, protein homology modeling demonstrated that genes within the same cluster share similar secondary and three-dimensional protein structures, whereas those from different subfamilies display a range of structural profiles. This conservation and diversity within subfamilies is consistent with the outcomes of phylogenetic analysis and classification. The structural variations among PsTPSs are likely to contribute to the functional diversity within the TPS gene family.
Our study also identified two gene pairs within the pea TPS gene families, indicative of gene duplication events, with both pairs being segmental duplicates. This suggests that the TPS gene family in peas may have expanded primarily through segmental duplication mechanisms. Homologous genes between peas and other species were localized to specific chromosomes, namely Ps1, Ps2, Ps4, Ps5, and Ps7, which have been conserved throughout the evolutionary history of peas. These findings suggest that changes in gene structure, potentially due to the gain or loss of exons or introns, could lead to functional differences among TPS genes [18]. This insight into the evolutionary trajectory and structural plasticity of TPS genes in peas contributes to our understanding of their potential roles in adaptation and diversification.
The regulatory roles of the cis-elements of the PsTPS gene family are central to the multifaceted processes of plant growth, development, and adaptation to environmental conditions. Examination of the promoter regions of the PsTPS genes predicted the presence of various cis-acting elements, which can be broadly divided into three categories: developmental response elements, hormone response elements, and stress response elements. Among them, elements that respond to light were the most abundant, highlighting the critical regulatory influence of light on gene expression. The characteristics of light, such as quality, intensity, and duration, exert a substantial impact on plant physiological processes such as photomorphogenesis, flowering, fruit pigmentation, and secondary metabolism [19,20]. Moreover, light has been established as influencing plant responses to various biotic and abiotic stresses, including photoinhibition and the management of stress response pathways [21]. A comprehensive understanding of the light-responsive elements and their interactions with other signaling pathways is essential for promoting plant growth, development, and stress recovery.
As is well documented, hormone response elements play a cardinal role in the hormonal signaling system of plants, located in the regulatory regions of target genes and interacting with transcription factors activated by plant hormones [22]. These interactions modulate gene expression which is critical for the plant growth and development processes regulated by hormones. Stress response elements are implicated in the adaptive mechanisms of plants to abiotic stresses such as drought and salinity, which can markedly affect the productivity of economically valuable plants [23]. The PsTPS gene family includes members with cis-acting regulatory elements associated with the response of plants to abiotic stress, playing a crucial role in environmental adaptation by triggering the necessary physiological and molecular responses to mitigate stress [24,25]. The distribution and proportion of these cis-regulatory elements vary among the PsTPS subfamilies, suggesting functional specialization.
Transcriptome technology was used to examine the expression profiles of PsTPS genes under various abiotic stress conditions. The analysis revealed that members of the same subfamily exhibited unique expression profiles, indicative of functional diversification. Under simulated drought conditions, significant differences in the expression levels of the 14 monitored PsTPS genes were observed at multiple time points compared to the baseline (0 h). The expression levels of the two genes also displayed significant changes at different time points following the salt stress treatment compared to the control group (0 h). These findings collectively imply that PsTPS genes are integral to the regulatory network that mediates the plant’s response to drought and salt stress.
The exploration of the PsTPS gene family yielded valuable insights into the genetic mechanisms employed by plants to adapt to stress. Leveraging this understanding is crucial for the strategic modification of these genes, with the aim of developing crop varieties that possess superior resilience to environmental stressors. Exploring the PsTPS gene family uncovered potential targets for genetic enhancement, thereby paving the way for crops that can withstand the challenges posed by adverse environmental conditions. This targeted approach to crop improvement is anticipated to significantly bolster agricultural productivity and sustainability amidst climate variability and other environmental challenges.
5. Conclusions
This study comprehensively assessed the PsTPS gene family in peas and identified 20 genes characterized by unique phylogenetic connections, gene structures, and regulatory elements. In addition, their transcriptional responses to abiotic stressors, such as drought and salinity, were investigated, highlighting their possible contributions to stress tolerance mechanisms. This in-depth profiling of the PsTPS gene family not only enriches our understanding of their physiological roles but also opens up avenues for enhancing the stress resistance of peas through selective breeding. Such advancements are expected to improve agricultural sustainability, particularly in the face of growing environmental adversities.
Conceptualization, N.L., Y.G. and H.Y.; methodology, H.Y. and B.L.; investigation, G.Z., Z.F., B.W., Y.B., Y.X. and Z.S.; writing—original draft preparation, H.Y.; writing—review and editing, N.L., Y.G. and H.Y.; project administration, N.L. All authors have read and agreed to the published version of the manuscript.
The original contributions presented in the study are included in the article/
The authors declare no competing interests. All analyzed data during this study are included in this published article and its
Footnotes
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Enlarge this image.Figure 1. Chromosomal locations of the PsTPS genes on the seven pea chromosomes. The distribution of PsTPS genes is relatively sparse, and they are not distributed on every chromosome. The highest distribution of PsTPS genes is observed on Chr5, which contains seven genes.
Enlarge this image.Figure 2. Phylogenetic tree incorporating TPS proteins from Pisum sativum L, Arabidopsis, and Glycine max. The tree of the TPS gene family was constructed by the IQ-TREE 2 software (Version 2.2.0) using the maximum likelihood (ML) method with 1000 bootstrap replicates. The color of the outer ring and branches denote TPS subfamilies.
Enlarge this image.Figure 3. The phylogenetic relationship, conserved motifs, and gene structure of PsTPSs. (a) The maximum likelihood (ML) phylogenetic tree of PsTPS proteins was constructed using a full-length sequence with 1000 bootstrap replicates; (b) Distribution of conserved motifs in PsTPS proteins. A total of 10 motifs were predicted, and the scale bar represents 100 aa; (c) Distribution of the TPS domain in PsTPSs; (d) The gene structures of PsTPSs, including introns (black lines) and exons (green rectangles). The scale bar indicates 1000 bp.
Enlarge this image.Figure 4. CREs on the putative promoters of PsTPSs. (a) Distribution of CREs identified in the 2000 bp upstream promoter region of PsTPSs; (b) The number of CREs on the putative promoters of PsTPSs. Numbers in the heatmap represent the number of elements.
Enlarge this image.Figure 5. Syntenic analyses of TPS genes in Pisum sativum, Arabidopsis, G. max. (a) Seven chromosomes from Pisum sativum (Ps1–Ps7) are mapped, with chromosome length expressed as Mb. Lines denote syntenic TPS gene pairs on the chromosomes. (b) The seven chromosomes of Pisum sativum (Ps1–7), five chromosomes of A. thaliana (At1–5), and twenty chromosomes of G. max (Gm1–20) are mapped. Lines represent syntenic TPS gene pairs.
Enlarge this image.Figure 6. Predicted protein–protein interaction networks of PsTPS proteins with other proteins using the STRING tool. Interactions between proteins are represented by gray lines.
Enlarge this image.Figure 7. Expression profiles of the eight PsTPS genes. The color scale from blue to red indicates increasing log2-transformed FPKM values.
Enlarge this image.Figure 8. Transcriptome analysis describes the expression levels of 14 PsTPS genes in peas under drought stress induced by a 20% concentration of PEG6000 solution and salt stress induced by a 300 mM concentration of NaCl solution. Each experiment was conducted independently with at least three replicates. ‘CK_0h’ represents the control group.
Supplementary Materials
The following supporting information can be downloaded at
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