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Article

Genome-Wide Identification of G3BP Family in U’s Triangle Brassica Species and Analysis of Its Expression in B. napus

by
Alain Tseke Inkabanga
1,2,†,
Qiheng Zhang
1,2,†,
Shanshan Wang
1,2,
Yanni Li
1,2,
Jingyi Chen
1,2,
Li Huang
1,2,
Xiang Li
1,2,
Zihan Deng
1,2,
Xiao Yang
1,2,
Mengxin Luo
1,2,
Lingxia Peng
1,2,
Keran Ren
1,2,
Yourong Chai
1,2,* and
Yufei Xue
1,2,*
1
Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
2
Engineering Research Center of South Upland Agriculture, Ministry of Education, Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2025, 14(14), 2247; https://doi.org/10.3390/plants14142247
Submission received: 17 June 2025 / Revised: 18 July 2025 / Accepted: 18 July 2025 / Published: 21 July 2025
(This article belongs to the Special Issue Plant Genetic Diversity and Molecular Evolution)
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Abstract

The RasGAP SH3 domain binding protein (G3BP) is a highly conserved family of proteins in eukaryotic organisms that coordinates signal transduction and post-transcriptional gene regulation and functions in the formation of stress granules. G3BPs have important roles in abiotic/biotic stresses in mammals, and recent research suggests that they have similar functions in higher plants. Brassica contains many important oilseeds, vegetables, and ornamental plants, but there are no reports on the G3BP family in Brassica species. In this study, we identified G3BP family genes from six species of the U’s triangle (B. rapa, B. oleracea, B. nigra, B. napus, B. juncea, and B. carinata) at the genome-wide level. We then analyzed their gene structure, protein motifs, gene duplication type, phylogeny, subcellular localization, SSR loci, and upstream miRNAs. Based on transcriptome data, we analyzed the expression patterns of B. napus G3BP (BnaG3BP) genes in various tissues/organs in response to Sclerotinia disease, blackleg disease, powdery mildew, dehydration, drought, heat, cold, and ABA treatments, and its involvement in seed traits including germination, α-linolenic acid content, oil content, and yellow seed. Several BnaG3BP DEGs might be regulated by BnaTT1. The qRT-PCR assay validated the inducibility of two cold-responsive BnaG3BP DEGs. This study will enrich the systematic understanding of Brassica G3BP family genes and lay a molecular basis for the application of BnaG3BP genes in stress tolerance, disease resistance, and quality improvement in rapeseed.

1. Introduction

RasGAPase-activating proteins (SH3 structural domain) binding proteins (G3BPs, also known as Rasputins) are a family of highly conserved RNA-binding proteins that developed during eukaryotic evolution [1]. G3BP is an important component of stress granules (SGs); SGs are generated in the cytoplasm in response to stresses, and might function in the regulation of mRNA metabolism in stress [2]. G3BPs contain four distinct structural domains [3]: a nuclear transporter factor 2 (NTF2)-like region, an acidic and proline-rich region, an RNA recognition motif (RRM), and an arginine- and glycine-rich region (RGG). Currently, there has been very few reports on plant G3BPs, unlike in mammals and Drosophila. Like those in mammals, plant G3BPs are also likely to participate in various cellular processes in which they coordinate signal transduction and post-transcriptional regulation, and meanwhile they can affect the formation of SGs.
G3BPs play important roles in the response to environmental stresses [3]. Based on the coexistence of conserved NTF2-like and RRM domains, eight G3BP family genes (AtG3BPs) were identified in Arabidopsis thaliana [1]. A gene expression analysis of AtG3BPs in response to different stresses, including abiotic stress, biotic stress, inducers, hormones, and nutrient starvation, revealed that all abiotic stresses induce gene expression, except oxidative stress, which inhibits gene expression [3]. For example, AtG3BP1, AtG3BP2, AtG3BP3, AtG3BP5, and AtG3BP7 are induced by cold stress. The necrotrophic fungus Botrytis cinerea induces the expression of AtG3BP3 and ATG3BP7; Pseudomonas syringae induces the expression of AtG3BP3 and represses the expression of AtG3BP2 [3]. Gibberellic acid (GA) induces the expression of AtG3BP2 and AtG3BP3 but suppresses AtG3BP7 expression, and abscisic acid (ABA) induces the expression of AtG3BP3 and AtG3BP5 [3]. Now there is no report on the involvement of G3BPs in seed traits, including seed oil content, seed fatty acid composition, yellow seed, seed germination, and seed development.
Rapeseed (Brassica napus, oilseed rape) is one of the most important oilseed crops worldwide, which provides about 13–16% of edible vegetable oil for humans [4]. Brassica is one of the most economically valuable genera in the Brassicaceae, with a wide variety of plants and with many subspecies, varieties, and cultivars, which have high agronomic and economic importance [5,6,7]. A. thaliana is closely related to Brassica species. Currently, only A. thaliana G3BPs have been identified at a whole-genome scale and have been reported to play potential roles in response to abiotic/biotic stresses [1,3]. The G3BP family genes in other plants including Brassica species have no genome-wide or function studies. In this study, the G3BP family genes from six U’s triangle Brassica species including B. rapa, B. oleracea, B. nigra, B. napus, B. juncea, and B. caritana are systematically identified at the whole-genome level. Based on transcriptome data, the expression patterns of B. napus G3BP (BnaG3BP) genes in various tissues/organs in response to Sclerotinia disease, blackleg disease, powdery mildew, dehydration, drought, heat, cold, and ABA treatments, and their involvement in seed oil content, seed fatty acid composition, and yellow seed traits, were analyzed. This will provide an important molecular basis for further study of the biological function of G3BPs in Brassica species, and for the application of BnaG3BP genes in stress tolerance, disease resistance, and quality improvement in rapeseed.

2. Results

2.1. Identification of G3BPs in U’s Triangle Brassica Species

A total of 27, 23, and 20 G3BP family genes were identified in the allotetraploid Brassica species, B. napus, B. juncea, and B. caritana, respectively (Table S1). There existed a total of 13, 13, and 10 G3BP family genes in the diploid progenitors, B. rapa, B. oleracea, and B. nigra, respectively. Here, eight G3BP family genes were also identified in A. thaliana, which is consistent with previously reported findings [1]. The gene sequences of the G3BP genes in these seven species ranged from 1564 bp to 8479 bp in length, whereas their coding regions varied from 1188 bp to 2628 bp. In addition, the G3BP proteins in these seven species ranged from 395 aa to 875 aa in length, their theoretical molecular weight varied from 43.11 kDa to 83.68 kDa, and their theoretical pI values ranged from 4.74 to 9.74. The subcellular localization prediction showed that the G3BP proteins of these seven species were mainly located in the chloroplasts, cytoplasm, mitochondria, nucleus, and vacuoles.

2.2. Phylogenetic Relationship Analysis

Phylogenetic analyses showed that the 114 G3BP family members from seven species were grouped into six clades based on sequence similarity: I (34), II (17), III (10), IV (24), V (19), and VI (10) (Figure 1). The number of each clade in these seven species is shown in detail in Table 1. For the eight G3BP genes in A. thalina, only the AT3G07250 ortholog was absent in the six U’s triangle Brassica species, which might support the hypothesis that AT3G07250 gene loss occurred in Brassica species after their separation from A. thalina in the evolutionary process.

2.3. Analysis of Gene Structure and Conserved Motifs

The gene structures and protein motifs of the G3BP family genes in Brassica species and A. thaliana were analyzed using GSDS 2.0 and the MEME online database, respectively. The results showed that there existed 3–14 introns in the G3BP genes in these seven species (Figure 2). The G3BP proteins from these seven species contained 10 protein conserved motifs (Motifs 1–10), and they were highly conserved in each G3BP clade.

2.4. Chromosome Location, Gene Duplication, and Selection Pressure Analysis

In total, 23 BnaG3BPs were unevenly located in the 10 chromosomes of B. napus, and the remaining 4 BnaG3BPs were mapped in unknown sites (Table S1). A total of 23 BjuG3BPs were unevenly distributed in the 12 chromosomes of B. juncea. There existed 19 BcaG3BPs that were unevenly distributed in the 10 chromosomes of B. carinata and 1 BcaG3BP in an unknown site. In both B. rapa and B. oleracea, 13 BraG3BPs and 13 BolG3BPs were unevenly mapped in five chromosomes. In B. nigra, 10 BniG3BPs were located in six chromosomes. The gene duplication analysis showed that there existed a total of 10 duplicated gene pairs for G3BPs in diploid Brassica species, which contained eight segmental duplications and two tandem duplications (Table S2). For G3BPs in allotetraploid Brassica species, a total of 75 duplicated gene pairs were identified, which included 68 segmental duplications and 7 tandem duplications. For these 85 duplicated gene pairs in the G3BPs of six Brassica species, all Ka/Ks ratios were below 1, suggesting that purifying selection might occur in the evolution process.

2.5. Prediction of Upstream miRNAs

The function of miRNAs lies in regulating the post-transcription expression of genes, and thus we predicted the miRNAs targeting G3BPs in six Brassica species and A. thaliana using the psRNATarget online database. As illustrated in Table S3, a total of 96 miRNAs were identified to target 63 G3BPs from these seven species, which primarily inhibit mRNA expression through a cleavage effect (96.87%). The mtr-miR2673a and mtr-miR2673b both were predicted to target 34 G3BPs in seven species, whereas the remaining miRNAs were predicted to target 1 to 10 G3BPs in seven species.

2.6. Analysis of SSR Loci

To analyze the distributions of SSR loci in G3BP genes from six Brassica species, we identified their SSR motifs using the online MISA-web database. As shown in Table S4, a total of 21 SSR loci were identified in the G3BP genes in six Brassica species and A. thaliana, which included seven types: mono- (p1, 2), di- (p2, 5), tri- (p3, 3), tetra- (p4, 4), penta- (p5, 3), and nonanucleotide (p9, 2), as well as compound type (c, 2). There existed nine SSR loci in seven BnaG3BPs, two in two BjuG3BPs, four in three BcaG3BPs, one in one BraG3BP, three in three BolG3BPs, and one in one BniG3BP.

2.7. Expression Patterns of BnaG3BP Genes in Various Tissues/Organs

To examine the organ specificity of BnaG3BP expression, based on the BrassicaEDB database, we analyzed the expression patterns of BnaG3BPs in 103 tissues and organs of the seven growth and developmental periods of B. napus. As shown in Figure 3 and Table S5, there was almost no expression in 103 tissues for BnaA07g24350D, BnaC06g25450D, BnaA07g24340D, BnaC06g06690D, and BnaC06g25440D. The BnaA07g24350D FPKM values were 1–1.91 only in seven tissues, BnaC06g25450D FPKM values were 1–3.71 only in eight tissues, BnaA07g24340D FPKM values were less than 0.75, and FPKM values of BnaC06g06690D and BnaC06g25440D were almost less than 0.05. The rest of the BnaG3BP genes were expressed in most tissues and organs (FPKM > 1), for example, the BnaC09g17770D FPKM values ranged from 2.18 to 77.68 in 103 tissues (Figure 3). BnaG3BP genes from clade III and V both had higher expression levels in various organs, while those from clade II and VI both had very lower expression levels. Some of BnaG3BPs in Clade I and IV possessed higher expressions and their remaining genes were expressed at lower levels.

2.8. Expression Patterns of BnaG3BP Genes in Response to Biotic Stresses

To detect the potential roles of BnaG3BP in plant disease defense, we analyzed their expression patterns in response to biotic stresses including Sclerotium stem rot (Sclerotium sclerotiorum), blackleg disease (Leptosphaeria maculans), and powdery mildew (Erysiphe cruciferarum), based on RNA-seq data (Tables S6–S8).
In response to S. sclerotiorum, there were six differentially expressed genes (DEGs) of BnaG3BP (BnaA02g14660D, BnaA06g39850D, BnaA07g27780D, BnaC06g30420D, BnaC07g22440D, and BnaCnng26260D) in a susceptible host (Westar), all of which were up-regulated (Figure 4). There were nine BnaG3BP DEGs in a resistant host (ZY821), of which seven (BnaA02g14660D, BnaA06g39850D, BnaA07g27780D, BnaC06g30420D, BnaC07g22440D, BnaC09g17770D, and BnaCnng26260D) were up-regulated, and two (BnaA03g39880D and BnaC07g30950D) were down-regulated. There were six commonly up-regulated BnaG3BP DEGs in both resistant and susceptible hosts. There was no commonly down-regulated DEGs. There existed three BnaG3BP DEGs that were only in the resistant host, of which one was up-regulated and two were down-regulated. There was no BnaG3BP DEG that was only in the susceptible host.
In the response of the susceptible host to L. maculans, there were no BnaG3BP DEGs 3 days after inoculation, and there were five BnaG3BP DEGs 7 days after inoculation, of which three were up-regulated (BnaAnng06690D, BnaC07g17480D, and BnaC07g30950D) and two were down-regulated (BnaA06g08750D and BnaC07g22440D).There were seven BnaG3BP DEGs 11 days after inoculation, of which one was up-regulated (BnaC07g17480D) and six were down-regulated (BnaA07g05030D, BnaA06g39850D, BnaC05g10060D, BnaA06g08750D, BnaC07g22440D, and BnaC09g05100D). In the response of the tolerant host to L. maculans, there were two BnaG3BP DEGs three days after inoculation, of which one was up-regulated (BnaAnng06690D), and one was down-regulated (BnaC07g22440D). There were three BnaG3BP DEGs (BnaAnng06690D, BnaC07g17480D, and BnaC02g41910D) seven days after inoculation, of which all were up-regulated. There were five BnaG3BP DEGs 11 days after inoculation, of which four were up-regulated (BnaAnng06690D, BnaC07g17480D, BnaA07g27780D, and BnaC09g17770D) and one was down-regulated (BnaC09g05100D) (Figure 5).
In response to E. cruciferarum, the FPKM values of a total of three BnaG3BPs were obtained from the transcriptome data. After inoculation with powdery mildew, R (resistant) vs. S (susceptible) had two BnaG3BP DEGs, of which BnaC05g10060D was up-regulated and BnaC07g17480D was down-regulated (Figure 6).

2.9. Expression Patterns of BnaG3BP Genes in Response to Abiotic Stresses

To examine the roles of BnaG3BP genes in response to abiotic stresses, we analyzed their expression patterns under cold, salt, dehydration, heat, drought, and ABA treatments based on RNA-seq data (Tables S9 and S10). Under dehydration stress, there were four BnaG3BP DEGs at 1 h after treatment, of which two were up-regulated (BnaC09g17770D and BnaA03g39880D) and two were down-regulated (BnaC07g22440D and BnaC09g05100D); there were two BnaG3BP DEGs for 8 h of treatment, in which one was up-regulated (BnaA03g39880D) and one was down-regulated (BnaC07g22440D) (Figure 7). Under NaCl stress, there were three BnaG3BP DEGs (BnaA07g05030D, BnaC07g22440D, and BnaC07g30950D), in which all were down-regulated at 4 h of treatment; there were four BnaG3BP DEGs (BnaC06g30420D, BnaC02g41910D, BnaA03g39880D, and BnaC09g05100D), of which all were up-regulated. Under ABA treatment, there existed two BnaG3BP DEGs (BnaC07g22440D and BnaC07g30950D) at 4 h, of which all were down-regulated; there were two BnaG3BP DEGs at 24 h, with one being up-regulated (BnaA03g39880D) and the other down-regulated (BnaC07g22440D). Under cold stress, there were three BnaG3BP DEGs, in which one gene was up-regulated (BnaA03g39880D) and two were down-regulated (BnaC07g22440D and BnaC07g30950D) after 4 h of treatment. There were seven BnaG3BP DEGs at 24 h of cold treatment, of which four were up-regulated (BnaC06g30420D, BnaA07g27780D, BnaA07g05030D, and BnaA06g08750D) and three were down-regulated (BnaC07g22440D, BnaC07g30950D, and BnaC09g05100D). Under heat stress, there were two BnaG3BP DEGs (BnaAnng06690D and BnaC07g17480D), which were both up-regulated (Figure 8). Under drought stress, there existed two BnaG3BP DEGs (BnaC07g30950D and BnaC05g10060D), which were both down-regulated.

2.10. Expression Patterns of BnaG3BP Genes Involved in Seed Germination Traits

To investigate the potential roles of BnaG3BPs in regulating seed germination traits, we analyzed their expression patterns during the seed germination process of three selected accessions (low, medium, or high germination rates; C032, C033, and C129, respectively) at 0, 12, 24, 48, and 72 h after imbibition, based on RNA-seq data (Table S11 and Figure 9). In total, there existed 14, 11, and 17 BnaG3BP DEGs in the seed germination process of C032, C033, and C129, respectively, in which there were 3 common BnaG3BP DEGs. In the C032, C033, and C129 accessions, there existed one, two, and four down-regulated BnaG3BP DEGs, respectively. There were 13, 9, and 13 up-regulated BnaG3BP DEGs in the C032, C033, and C129 accessions, respectively. This result shows that these BnaG3BP DEGs might be involved in the regulation of seed germination traits.

2.11. Expression Patterns of BnaG3BP Genes Involved in Yellow Seed Trait

To examine the potential roles of BnaG3BPs in yellow seed traits, we analyzed their expression patterns in coats and embryos from black seeds and yellow seeds at 35, 40, and 46 DAF in rapeseed (Table S12 and Figure 10). In total, there were two BnaG3BP DEGs (BnaA06g08750D and BnaA02g14660D, both down-regulated) in the yellow coats vs. the black coats, whereas three BnaG3BP DEGs were observed in the yellow embryos vs. the black embryos, of which one was down-regulated (BnaA06g08750D) and two were up-regulated (BnaA07g05030D and BnaA09g05550D). This result suggests that these BnaG3BP DEGs might play potential roles in the regulation of yellow seed traits.

2.12. Expression Patterns of BnaG3BP Genes Involved in Seed ALA Trait

To investigate the potential function of BnaG3BPs in regulating seed linolenic acid traits, we analyzed their expression patterns in 24-day-old seed embryos from two rapeseed inbred lines with high (YH25005 and R8Q10) and low (SW and A28) α-linolenic acid (ALA) contents (Table S13). We found that there were 0, 16, 1, and 10 BnaG3BP DEGs in R8Q10 vs. A28, YH25005 vs. A28, R8Q10 vs. SW, and YH5005 vs. SW, respectively (Figure 11), in which only the common BnaG3BP DEG (BnaA06g08750D) in R8Q10 vs. SW and YH5005 vs. SW was up-regulated, and the rest of the BnaG3BP DEGs all were down-regulated. This suggests that the BnG3BP DEGs are likely to mainly be negative regulators of seed ALA content.

2.13. Expression Patterns of BnaG3BP Genes Involved in Seed Oil Content Traits

To determine the potential roles of BnaG3BPs in regulating seed oil content, we analyzed their expression patterns in 7-, 10-, 14- and 45-day-old seeds from two rapeseed cultivars with high (ZS11)/low (ZY821) seed oil contents (Table S14 and Figure 12). In total, there were eight BnaG3BP DEGs (two down-regulated and six up-regulated) in ZS11_7d vs. ZY821_7d. There existed six BnaG3BP DEGs (three down-regulated and three up-regulated) in ZS11_10d vs. ZY821_10d. There were five BnaG3BP DEGs (one down-regulated and four up-regulated) in ZS11_14d vs. ZY821_14d. There existed six BnaG3BP DEGs (one down-regulated and five up-regulated) in ZS11_45d vs. ZY821_45d. Several genes kept the same differential expression trends along the seed development process, e.g., BnaC09g05100D, BnaC07g26640D, BnaA07g05030D, BnaA06g39850D, BnaA06g08750D and BnaAnng06690D were successively higher in high-oil ZS11 than in low-oil ZY821, while BnaA06g36440D, BnaA09g16820D and BnaC07g17480D were successively lower in ZS11 than in ZY821.This result shows that these BnaG3BP DEGs might be involved in the regulation of seed oil content.

2.14. Expression Patterns of BnaG3BP Genes in BnaTT1 Transgenic Lines

TT1s negatively regulate seed oil accumulation but positively regulate flavonoid-proanthocyanidin biosynthesis [8]. To detect whether BnaG3BPs are target genes of B. napus TT1 (BnaTT1), we analyzed BnaG3BP DEGs in the transcriptome data of the BnaTT1 transgenic lines in our group (unpublished). In total, there existed one and three BnaG3BP DEGs in the mid-stage seeds of transgenic rapeseeds over-expressing pNapA::BnaTT1 (NOE) and pBAN::BnaTT1 (BOE), respectively. As shown in Table 2 and Table 3 (reference source data unpublished), after the overexpression of NOE and BOE, these four BnaG3BP DEGs (BnaC05g10060D, BnaA03g39880D, BnaC07g17480D, and BnaC07g30950D) all were down-regulated, and BnaC07g17480D DEG was specifically expressed in the mid-stage seed coat, especially in the inner integument (Figure S1). This suggests that they might be downstream target genes of BnaTT1 and might be involved in the regulation of seed development and metabolism.

2.15. Validation of Selected Cold-Responsive Genes

To validate the accuracy of the transcriptome data, a qRT-PCR analysis was conducted on two representative cold-responsive BnaG3BP DEGs. The results showed that these two DEGs were both significantly up-regulated under cold treatment (Figure 13), which is consistent with their RNA-seq results. Additionally, the chlorophyll fluorescence parameters of rapeseed leaves after cold treatment also changed significantly. The NPQ values were up-regulated and the ΦPSII values were down-regulated, suggesting their potential application in cold stress detection at an early-stage.

3. Discussion

Substantial studies have showed that RNA-binding proteins (RBPs) play important roles in regulating post-transcriptional changes, which is of great significance for plant growth and development, and defenses against biotic/abiotic stresses [1,9,10,11]. G3BPs belong to a highly conserved family of RBPs in eukaryotic organisms, which function in signal transduction and post-transcriptional gene regulation, and play important roles in the formation of SGs [1,2,3]. Unlike mammals and Drosophila, there are limited reports on the function plant G3BPs as well as their roles in the production of SGs [3]. In the TAIR10 reference genome of A. thaliana, it had been reported that there exists eight G3BP family members [1]. Here, we further validated that there are also the same number of AtG3BPs in the TAIR11 genome. According to the U’s triangle model of Brassica species, based on natural hybridization, the diploid ancestors B. rapa (AA) and B. oleracea (CC) putatively produce the allotetraploid B. napus (AACC), diploid parents B. rapa (AA) and B. nigra (BB) putatively generate the allotetraploid B. juncea (AABB), and diploid progenitors B. nigra (BB) and B. oleracea (CC) putatively give rise to B. caritana (BBCC) [12,13,14]. In this study, the allotetraploids B. napus, B. juncea, and B. caritana contained 27, 23, and 20 G3BP genes, respectively, and a total of 13, 13, and 10 G3BP genes were identified in the diploid progenitors B. rapa, B. oleracea, and B. nigra, respectively. Obviously, the total number of members of the G3BP family in the three diploid progenitors and three allotetraploid Brassica species basically conform to U’s triangle model. However, the total numbers of the G3BP family genes from these six Brassica species of U’s triangle still need to be further determined when their gap-free T2T pan-genomes all are published.
In A. thaliana, the expression profiles of the AtG3BP genes were affected by bacterial, fungal, and different abiotic stresses and hormones, suggesting that AtG3BPs have potential roles in various stress signaling pathways [3]. In B. napus, we also identified a variety of BnaG3BP DEGs in responses to Sclerotinia disease, blackleg disease, and powdery mildew, suggesting they might be involved in disease resistance by post-transcriptional gene regulation. Moreover, BnaG3BP DEGs were identified after treatments of various abiotic stresses including heat, drought, dehydration, NaCl, cold, and ABA, suggesting these BnaG3BP DEGs might play potentially important roles in response to abiotic stresses. These results will provide insights for the potential application of BnaG3BP genes in the genetic improvement of disease resistance and abiotic stress tolerance in B. napus.
Currently, there has been no report on the involvement of G3BPs in improvements of seed traits including seed germination, seed oil content, seed ALA content, and yellow seed traits. As for RBPs, there were several literatures regarding the regulation of traits including seed germination, seed development, and seed size. For example, in rice and wheat, EOG1, which encodes an RNA-binding protein with two RRMs and one SPOC domain, negatively regulates seed size and weight [15]. In A. thaliana, the plant-specific SCL30a gene encodes an RNA-binding protein belonging to splicing regulators and it was found that SCL30a affected ABA-related seed traits and retarded germination [16]. In this study, we found that there exists a large number of BnaG3BP DEGs that are involved in seed germination, seed oil content, seed ALA content, and yellow seed traits. G3BPs belong to one type of RNA-binding proteins, and thus encoding proteins of these identified BnaG3BP DEGs that may impact seed traits might function via post-transcriptional mechanisms such as pre-mRNA processing, alternative splicing, mRNA stabilization, RNA editing, and mRNA transport [11].
TT1 negatively regulates oil accumulation but positively regulates flavonoid-proanthocyanidin biosynthesis [8]. In this study, there existed many BnaG3BP DEGs in the pairwise transcriptome comparisons of BOE vs. WT and NOE vs. WT for the mid-stage seeds of BnaTT1 overexpression plants, suggesting that these BnaG3BP DEGs might be target genes for the BnaTT1 transcription factor, and might be involved in seed development and seed metabolism.

4. Materials and Methods

4.1. Acquisition of the Reference Genomes of Brassica Species and A. thaliana

The reference genomes of B. napus (Brana_Dar_V5), B. rapa (Brara_Chiifu_V3.0), B. juncea (Braju_AU213_V1.0), and B. nigra (Brani_Ni100_V2) were downloaded from BRAD database (http://brassicadb.cn/, accessed on 1 March 2023). The reference genomes of B. oleracea (BOL), B. caritana, and A. thaliana (v11) were downloaded from EnsemblPlants database (http://plants.ensembl.org/index.html, accessed on 23 February 2023), Brassica genomics database (http://bio2db.com, accessed on 26 March 2023), and TAIR database (http://www.arabidopsis.org/, accessed on 23 February 2023), respectively.

4.2. Identification of G3BP Gene Family in Brassica Species and A. thaliana

Since G3BP proteins have NTF2 (PF02136) and RRM_1 (PF00076) conserved domains, hmmsearch was performed to retrieve candidate G3BP family genes from seven species, including Brassica species and A. thaliana, by using the HMMER 3.0 software with NTF2 and NTF2 domains as the bait, respectively. Subsequently, proteins with both PF02136 and PF00076 conserved domains were retained by manual checking as well as validation against the SMART database (http://smart.embl-heidelberg.de/, accessed on 31 March 2023); otherwise, they were considered as non-G3BP proteins, were deleted, and were not further analyzed.

4.3. Bioinformatics Analysis

Based on gene sequences and coding region sequences of G3BPs from Brassica species and A. thaliana, their exon–intron structural features were analyzed using the online website GSDS2.0 (http://gsds.cbi.pku.edu.cn/, accessed on 16 April 2023). The conserved motifs in their G3BP proteins were identified using the MEME suite 5.5.2 database (http://meme-suite.org/tools/meme, accessed on 16 April 2023) with default parameters. The theoretical isoelectric point (pI) and molecular weight (Mw) of their G3BP proteins were calculated using the ProtParam website (https://web.expasy.org/protparam/, accessed on 17 April 2023). Using the WoLF PSORT II database (https://www.genscript.com/tools/wolf-psort, accessed on 18 April 2023) and the Plant-mPLoc database (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 18 April 2023), subcellular localizations of their G3BP proteins were predicted. SSR motifs in their G3BP genes were predicted on MISA-web database (https://webblast.ipk-gatersleben.de/misa/, accessed on 20 March 2025). The upstream miRNA targeting their G3BP genes were predicted on psRNATarget according to the default parameters with the expected value of 3 (https://www.zhaolab.org/psRNATarget/, accessed on 20 March 2025).

4.4. Phylogenetic Analysis

The sequences of G3BP family proteins in seven species were multi-aligned using MAFFT7 software, and then neighbor-joining (NJ) phylogenetic tree was constructed using MEGA7 software in p-distance mode and their reliability was tested using bootstrap analysis (1000 replicates).

4.5. Expression Profiling Based on Transcriptome Data

The transcriptome data of 103 tissues and organs of rapeseed at seven growth and development stages were obtained from BrassicaEDB database [17] (https://brassica.biodb.org/, accessed on 15 April 2023). The NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 9 April 2023) provided transcriptome data of rapeseed in response to Sclerotinia stem pot (GSE81545) [18], blackleg (GSE77723) [19], and powdery mildew (GSE188377) [20] diseases, as well as heat and drought stresses (GSE156029), seed germination trait (GSE137230) [21], seed ALA content trait (GSE186952) [22], and yellow-seed trait (PRJNA597958) [23]. From the NGDC database (https://ngdc.cncb.ac.cn/, accessed on 14 September 2024), transcriptome data of rapeseed in response to dehydration, cold, salt, and ABA treatments (CRA001775) [24], and involvement in seed oil content (PRJCA001246) [25] were retrieved. BnaG3BP was considered as differentially expressed gene (DEG) if |FPKM/TPM| ≥ 1 and |log2FoldChange| ≥ 1. TBtools/TBtools-II [26] was used to perform cluster analysis and heatmap creation. Our group have obtained RNA-seq data for mid-stage seeds of transgenic rapeseed overexpressing BnaTT1 and the transcriptome data were mined to identify BnaG3BP DEGs.

4.6. The qRT-PCR Analysis

Total RNAs were extracted from the seedling leaves of rapeseed Westar cultivar at 0, 24, and 48 h under cold stress (4◦C), using the RNAsimple Total RNA Kit (DP419, Tiangen, Beijing, China). First-strand total cDNA was produced using the PrimeScript Reagent Kit with gDNA Eraser (RR047A, Takara, Dalian, China). The qRT-PCR assay was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (RR820A, Takara, Dalian, China) on a CFX Real-time PCR System (Bio-Rad, Irvine, CA, USA). Each reaction was performed with three replicates. Two representative cold-responsive BnaG3BP DEGs, BnaA07g05030D and BnaC06g30420D, were chosen to perform the qRT-PCR assay with 25sRNA as internal control (Table S15) as previously described by our group [27].

4.7. Chlorophyll Fluorescence Imaging

The fluorescence parameters and chlorophyll fluorescence images of the rapeseed seedling leaves at 0, 24, and 48 h under cold stress were analyzed on the plant phenotype imager (a chlorophyll fluorescence imaging system FluorCam7.0; Photon Systems Instruments, Brno, Czech Republic; device no. 20A00005).

5. Conclusions

In this study, a total of 114 G3BP genes were identified in six Brassica species of U’s triangle (B. napus, B. juncea, B. caritana, B. rapa, B. oleracea, and B. nigra) and A. thaliana. Their gene structure, protein motifs, gene duplication type, phylogeny, subcellular localization, SSR loci, and upstream miRNAs were then analyzed. Based on transcriptome data, we also analyzed the expression patterns of BnaG3BP genes in various tissues/organs and in response to Sclerotinia disease, blackleg disease, powdery mildew, dehydration, drought, heat, cold, and ABA treatments. The RNA-seq data showed that differential expression genes of BnaG3BP existed after the overexpression of BnaTT1 in rapeseed. The qRT-PCR assay validated the inducibility of two representative cold-responsive BnaGBP DEGs. This study will promote the systematic understanding of the G3BP family genes of U’s triangle Brassica species and lay a molecular foundation for the potential application of BnaG3BP DEGs in stress tolerance, disease resistance, and seed quality traits in rapeseed.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants14142247/s1. Table S1. Basic information of G3BP family genes in six Brassica species and A. thaliana; Table S2. Gene duplication types and Ka/Ks analysis for duplicated gene pairs of G3BP family genes in six Brassica species; Table S3. Basic information of predicted miRNAs targeting G3BPs in six Brassica species and A. thaliana; Table S4. Basic information of predicted SSR loci in G3BP family genes from six Brassica species and A. thaliana; Table S5. The FPKM values of BnaG3BP genes in various tissues/organs; Table S6. The FPKM values of BnaG3BP genes in response to Sclerotium sclerotiorum; Table S7. The FPKM values of BnaG3BP genes in response to Leptosphaeria maculans; Table S8. The FPKM values of BnaG3BP genes in response to Erysiphe cruciferarum; Table S9. The average FPKM values of BnaG3BP genes in response to multiple abiotic stresses; Table S10. The FPKM values of BnaG3BP genes in response to heat and drought; Table S11. The TPM values of BnaG3BP genes in seed germination processes; Table S12. The TPM values of BnaG3BP genes involved in the yellow seed trait; Table S13. The FPKM values of BnaG3BP genes involved in the seed ALA content trait; Table S14. The TPM values of BnaG3BP genes involved in seed oil content traits; Table S15. The qRT-PCR primers used in this study; Figure S1. The eFP heatmap (showing FPKM values) of BnaG3BP DEGs that might be regulated by BnaTT1 on BrassicaEDB database.

Author Contributions

Conceptualization, Y.C.; methodology, A.T.I. and X.Y.; software, Q.Z. and S.W.; validation, A.T.I. and Y.L.; formal analysis, Q.Z. and J.C.; investigation, L.H. and X.L.; resources, Y.C. and Y.X.; data curation, L.P. and K.R.; writing—original draft preparation, A.T.I. and Q.Z.; writing—review and editing, Y.X. and Y.C.; visualization, M.L. and Z.D.; supervision, Y.C. and Y.X. Y.X. undertook the use and maintenance of the plant phenotype imager. All authors have read and agreed to the published version of the manuscript.

Funding

The research was funded by the National Key R&D Program of China (2023YFF1000702), National Natural Science Foundation of China (32272105; 32001441; 31871549), Fundamental Research Funds for the Central Universities (SWU-KT25024), Experimental Technology Research Program of Southwest University (SYJ2025024), and Southwest University Training Program of Innovation and Entrepreneurship for Undergraduates (X202510635035).

Data Availability Statement

All additional datasets supporting the findings of this study are included within the article and Supplementary Materials.

Conflicts of Interest

All of the authors declare that there are no conflicts of interest.

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Plants 14 02247 g001
Figure 1. Phylogenetic tree of G3BP proteins among B. napus, B. juncea, B. caritana, B. rapa, B. oleracea, B. nigra, and A. thaliana.
Figure 1. Phylogenetic tree of G3BP proteins among B. napus, B. juncea, B. caritana, B. rapa, B. oleracea, B. nigra, and A. thaliana.
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Plants 14 02247 g002
Figure 2. Gene exon–intron structure (a) and protein conserved motifs (b) of G3BP family members in B. napus, B. juncea, B. caritana, B. rapa, B. oleracea, B. nigra, and A. thaliana.
Figure 2. Gene exon–intron structure (a) and protein conserved motifs (b) of G3BP family members in B. napus, B. juncea, B. caritana, B. rapa, B. oleracea, B. nigra, and A. thaliana.
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Plants 14 02247 g003
Figure 3. Expression patterns of BnaG3BP genes in various tissues/organs of B. napus. DAF, days after flowering; HAG, hours after germination.
Figure 3. Expression patterns of BnaG3BP genes in various tissues/organs of B. napus. DAF, days after flowering; HAG, hours after germination.
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Plants 14 02247 g004
Figure 4. Expression patterns of BnaG3BP genes in the leaves of susceptible (Westar) and tolerant (ZY821) genotypes of rapeseed infected with S. sclerotiorum at 24 h post-inoculation (24 hpi).
Figure 4. Expression patterns of BnaG3BP genes in the leaves of susceptible (Westar) and tolerant (ZY821) genotypes of rapeseed infected with S. sclerotiorum at 24 h post-inoculation (24 hpi).
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Plants 14 02247 g005
Figure 5. Expression patterns of BnaG3BP genes in the cotyledons of rapeseed susceptible (Westar) and resistant (DF78) lines at 0 day, 3 days, 7 days, and 11 days post-L. maculans inoculation.
Figure 5. Expression patterns of BnaG3BP genes in the cotyledons of rapeseed susceptible (Westar) and resistant (DF78) lines at 0 day, 3 days, 7 days, and 11 days post-L. maculans inoculation.
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Plants 14 02247 g006
Figure 6. Expression patterns of BnaG3BP genes in rapeseed susceptible (S) and resistant (R) lines after powdery mildew inoculation.
Figure 6. Expression patterns of BnaG3BP genes in rapeseed susceptible (S) and resistant (R) lines after powdery mildew inoculation.
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Plants 14 02247 g007
Figure 7. Expression patterns of BnaG3BP genes in 3-week-old rapeseed plants under multiple abiotic stresses. Dehydration (1 h and 8 h), ABA (25 μM; 4 h and 24 h), NaCl (200 mM; 4 h and 24 h), and cold (4 °C, 4 h and 24 h).
Figure 7. Expression patterns of BnaG3BP genes in 3-week-old rapeseed plants under multiple abiotic stresses. Dehydration (1 h and 8 h), ABA (25 μM; 4 h and 24 h), NaCl (200 mM; 4 h and 24 h), and cold (4 °C, 4 h and 24 h).
Plants 14 02247 g007
Plants 14 02247 g008
Figure 8. Expression patterns of BnaG3BP genes under heat and drought conditions. D3d, drought (withdrawing water) treated for 3 days. HT, heat treated at temperature of 40 °C for 3 h.
Figure 8. Expression patterns of BnaG3BP genes under heat and drought conditions. D3d, drought (withdrawing water) treated for 3 days. HT, heat treated at temperature of 40 °C for 3 h.
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Plants 14 02247 g009
Figure 9. Expression patterns of BnaG3BP genes in seed germination process of three selected accessions (low, medium or high germination rates; C032, C033 and C129, respectively) at 0, 12, 24, 48 and 72 h after imbibition.
Figure 9. Expression patterns of BnaG3BP genes in seed germination process of three selected accessions (low, medium or high germination rates; C032, C033 and C129, respectively) at 0, 12, 24, 48 and 72 h after imbibition.
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Plants 14 02247 g010
Figure 10. Expression patterns of BnaG3BP genes involved in yellow seed traits. Yellow seed coats at LC1 (35 days after flowering (DAF)), LC2 (40 DAF), and LC3 (46 DAF); yellow seed embryos at LE1 (35 DAF), LE2 (40 DAF), and LE3 (46 DAF); black seed coats at HC1 (35 DAF), HC2 (40 DAF), and HC3 (46 DAF); and black seed embryos a HE1 (35 DAF), HE2 (40 DAF), and HE3 (46 DAF).
Figure 10. Expression patterns of BnaG3BP genes involved in yellow seed traits. Yellow seed coats at LC1 (35 days after flowering (DAF)), LC2 (40 DAF), and LC3 (46 DAF); yellow seed embryos at LE1 (35 DAF), LE2 (40 DAF), and LE3 (46 DAF); black seed coats at HC1 (35 DAF), HC2 (40 DAF), and HC3 (46 DAF); and black seed embryos a HE1 (35 DAF), HE2 (40 DAF), and HE3 (46 DAF).
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Plants 14 02247 g011
Figure 11. Expression patterns of BnaG3BP genes in 24-day-old seed embryos from two rapeseed inbred lines with high (YH25005 and R8Q10)/low (SW and A28) α-linolenic acid traits.
Figure 11. Expression patterns of BnaG3BP genes in 24-day-old seed embryos from two rapeseed inbred lines with high (YH25005 and R8Q10)/low (SW and A28) α-linolenic acid traits.
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Plants 14 02247 g012
Figure 12. Expression patterns of BnaG3BP genes in 7-, 10-, 14- and 45-day-old seeds from two rapeseed cultivars with high (ZS11)/low (ZY821) seed oil content.
Figure 12. Expression patterns of BnaG3BP genes in 7-, 10-, 14- and 45-day-old seeds from two rapeseed cultivars with high (ZS11)/low (ZY821) seed oil content.
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Plants 14 02247 g013
Figure 13. Impact of cold stress on photosynthesis of rapeseed leaves (a,b), and qRT-PCR expression levels of selected cold-responsive BnaG3BP DEGs with 25sRNA as internal control (c). Standard images of Fv/Fm, ΦPSII, and NPQ from cold-treated rapeseed leaves at 0, 24, and 48 h. False color scale is used for each parameter. Values represent average ± SD of three biological replicates. Fv/Fm, maximum quantum yield of PSII; ΦPSII, effective quantum yield of PSII; and NPQ, non-photochemical quenching. * p < 0.05 and ** p < 0.01 compared with samples at 0 h.
Figure 13. Impact of cold stress on photosynthesis of rapeseed leaves (a,b), and qRT-PCR expression levels of selected cold-responsive BnaG3BP DEGs with 25sRNA as internal control (c). Standard images of Fv/Fm, ΦPSII, and NPQ from cold-treated rapeseed leaves at 0, 24, and 48 h. False color scale is used for each parameter. Values represent average ± SD of three biological replicates. Fv/Fm, maximum quantum yield of PSII; ΦPSII, effective quantum yield of PSII; and NPQ, non-photochemical quenching. * p < 0.05 and ** p < 0.01 compared with samples at 0 h.
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Table 1. The total number of G3BP genes in each clade of B. napus, B. juncea, B. caritana, B. rapa, B. oleracea, B. nigra, and A. thaliana.
Table 1. The total number of G3BP genes in each clade of B. napus, B. juncea, B. caritana, B. rapa, B. oleracea, B. nigra, and A. thaliana.
CategoryB. napusB. junceaB. caritanaB. rapaB. oleraceaB. nigraA. thaliana
Total2723201313108
I8774431
II5322212
III2221111
IV6543321
V4442221
VI2211112
Table 2. BnaG3BP DEG in transgenic rapeseeds (Westar cultivar) overexpressing pNapA::BnaTT1 (NOE, 20 days after pollination (DAP) seeds, seed embryo-specific promoter).
Table 2. BnaG3BP DEG in transgenic rapeseeds (Westar cultivar) overexpressing pNapA::BnaTT1 (NOE, 20 days after pollination (DAP) seeds, seed embryo-specific promoter).
Gene IDNOE_CountWT_Countlog2FCp Valuep Adj
BnaC05g10060D67.51207.70−1.624.25 × 10−85.58 × 10−7
Table 3. BnaG3BP DEGs in transgenic rapeseeds (Westar cultivar) overexpressing pBAN::BnaTT1 (BOE, 20 DAP seeds, seed coat-specific promoter).
Table 3. BnaG3BP DEGs in transgenic rapeseeds (Westar cultivar) overexpressing pBAN::BnaTT1 (BOE, 20 DAP seeds, seed coat-specific promoter).
Gene IDBOE_CountWT_Countlog2FCp Valuep Adj
BnaA03g39880D100.39205.50−1.030.000.01
BnaC07g17480D76.33172.32−1.170.000.01
BnaC07g30950D92.37190.97−1.050.000.01
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Inkabanga, A.T.; Zhang, Q.; Wang, S.; Li, Y.; Chen, J.; Huang, L.; Li, X.; Deng, Z.; Yang, X.; Luo, M.; et al. Genome-Wide Identification of G3BP Family in U’s Triangle Brassica Species and Analysis of Its Expression in B. napus. Plants 2025, 14, 2247. https://doi.org/10.3390/plants14142247

AMA Style

Inkabanga AT, Zhang Q, Wang S, Li Y, Chen J, Huang L, Li X, Deng Z, Yang X, Luo M, et al. Genome-Wide Identification of G3BP Family in U’s Triangle Brassica Species and Analysis of Its Expression in B. napus. Plants. 2025; 14(14):2247. https://doi.org/10.3390/plants14142247

Chicago/Turabian Style

Inkabanga, Alain Tseke, Qiheng Zhang, Shanshan Wang, Yanni Li, Jingyi Chen, Li Huang, Xiang Li, Zihan Deng, Xiao Yang, Mengxin Luo, and et al. 2025. "Genome-Wide Identification of G3BP Family in U’s Triangle Brassica Species and Analysis of Its Expression in B. napus" Plants 14, no. 14: 2247. https://doi.org/10.3390/plants14142247

APA Style

Inkabanga, A. T., Zhang, Q., Wang, S., Li, Y., Chen, J., Huang, L., Li, X., Deng, Z., Yang, X., Luo, M., Peng, L., Ren, K., Chai, Y., & Xue, Y. (2025). Genome-Wide Identification of G3BP Family in U’s Triangle Brassica Species and Analysis of Its Expression in B. napus. Plants, 14(14), 2247. https://doi.org/10.3390/plants14142247

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