https://orcid.org/0000-0003-4463-0218
Abstract
The GRAS transcription factor (TF) is a plant-specific regulator that plays a critical role in plant growth, development and response to various abiotic stresses. While the functions of many GRAS TFs have been extensively studied in numerous model plants with fully sequenced genomes, the GRAS TFs in white clover (Trifolium repens L.) remain elusive despite the sequencing of its genome. To bridge this knowledge gap, homologs of GRAS TFs from Arabidopsis were employed and subjected to a genome-wide blast against white clover proteins. This analysis identified and characterized 102 putative GRAS genes, designated as TrGRAS, based on the presence of GRAS domains in positive blast hits. Concurrently, an exploration of their gene structures, classification, evolutionary relationships and conservative motifs provided insights into the distinctive features of TrGRAS in white clover. Furthermore, gene duplication analysis revealed an expansion of TrGRAS genes in the PAT1 and LISCL subfamilies, indicating functional diversity within the white clover genome. Lastly, RNA-seq and quantitative reverse transcription-PCR (qRT-PCR) experiments confirmed their higher expression during the early or intermittent responses to cold stress, suggesting their potential and crucial roles in determining the cold tolerances of white clover. The analysis of TrGRAS genes, coupled with expression studies, contributes to the functional characterization of TrGRAS genes in white clover. This knowledge is pivotal for informing genetic improvement strategies in future breeding efforts aimed at enhancing cold tolerance in white clover.
Introduction
Plants have evolved diverse metabolic pathways to thrive in challenging environments, including cold, drought and salinity. Numerous genes and proteins actively participate in these intricate pathways [Citation1–3]. Notably, transcription factors (TFs) stand out among these genetic elements. TFs can bind to DNA regions (named cis-acting elements) and regulate the expression of downstream genes. Throughout their life cycle, plants have developed a multitude of TFs, such as AP2/ERF, MYB, WRKY and others, each uniquely equipped to manage various processes. These TFs play pivotal roles in both the growth processes of plants and their responses to diverse stresses [Citation1,Citation4].
The GRAS transcription factor (TF) earned its nomenclature from the original identification of three members: gibberellic acid insensitive (GAI) [Citation5], repressor of GA1 (RGA) [Citation6] and Scarecrow [Citation7]. Most GRAS TFs consist of 400–770 amino acids and feature a highly conserved domain known as the GRAS domain [Citation8]. As genome sequences of several model plants have been completed and released, extensive analyses of GRAS TFs have been conducted in plants such as Arabidopsis (Arabidopsis thaliana) [Citation9], rice (Oryza sativa L.) [Citation9], grape (Vitis vinifera L.) [Citation10], soybean (Glycine max) [Citation11] and tomato (Solanum lycopersicum) [Citation12]. These studies have revealed a high degree of divergence among GRAS TFs in different plants. For instance, Arabidopsis (A. thaliana) harbors eight subfamilies [Citation9], soybean (G. max) has nine subfamilies [Citation11], and eggplant (Solanum melongena L.) possesses thirteen GRAS subfamilies [Citation13]. The varied gene structures of GRAS TFs suggest their diverse functions across different plants, influencing processes such as seedling growth, tissue development and responses to various stresses [Citation8,Citation14]. Of particular note, DELLA proteins, a subgroup of GRAS TFs, have been implicated in the CBF-regulation pathway, a key determinant in plant cold tolerance [Citation15], such as SlGRAS4 directly bind to tomato SlCBF promoters to activate their transcription [Citation16]. Additionally, other GRAS TFs have been characterized for their pivotal roles in responding to cold stress. For instance, VaPAT1 enhances cold tolerance by regulating JA biosynthesis in Arabidopsis (A. thaliana) [Citation17] and grape (V. vinifera L.) [Citation18]. Xing et al. [Citation19] have found than increasement of GRAS24 expression would enhance postharvest chilling tolerance in tomato fruit. Furthermore, Li et al. [Citation20] reported that RsSHRc reliably functions in eliminating reactive oxygen species under chilling stress.
White clover (Trifolium repens L.) is a perennial legume plant with widespread distribution in temperate and cool-temperate regions [Citation21]. Recognized for its robust root development, prostrate growth, rapid regeneration, high yields and quality attributes, white clover holds significant economic importance. It serves as an ideal forage and a valuable plant for gardening purposes. However, over its lifespan of several years, white clover faces threats from various adverse conditions, including salt, drought and cold stress. Particularly in high-latitude regions, harsh winters pose a severe challenge, significantly limiting and endangering white clover [Citation22]. Addressing the need to enhance the cold tolerance of white clover has become a substantial challenge in both breeding and production. With the release of white clover genome sequences [Citation23], research efforts have delved into genome-wide analyses, focusing on functional genes, particularly transcription factors such as SPL and WRKY. These investigations contribute valuable insights to white clover genetic research and molecular breeding strategies [Citation24,Citation25].
In this study, we successfully identified 102 GRAS genes within the white clover genome. A comprehensive characterization of their molecular features, including gene structure, conservative domains, genomic duplication and promoter constituents, was undertaken. Furthermore, the gene expression levels of these GRAS genes were assessed using RNA-seq data, and their responses to cold stress were further validated through qRT-PCR experiments. These findings not only offer crucial insights into the evolution of these genes, their group classifications and molecular functions within the GRAS family, but also hold significant implications for genetic breeding research in white clover. The results, particularly in the context of understanding and improving cold tolerance, are poised to contribute to advancements in white clover breeding strategies in the future.
Materials and methods
Plant materials
Seeds of the white clover cultivar Haifa were generously provided by Chinese Barenbrug Ltd. Com. (Beijing, China). The germination and cultivation of white clover followed a previously described method, utilizing pots (0.5 L) filled with a perlite and sand mixture in a ratio of 3:1 (v/v) [Citation26]. Briefly, seeds were sown in pots (0.5 L), with approximately 10–15 plants per pot, and placed in an environmental growth chamber with a light intensity of 100 µmol/(m2·s) at 24/18 °C (day/night) under a 12-h photoperiod. The plants received irrigation with half-strength Hoagland solution at a rate of 20–30 mL per pot every two days. After four weeks of growth, all seedlings were subjected to a chilling treatment at 4 °C. Cold stress was initiated at 8:00 am and lasted until 8:00 am the following morning. These seedlings were randomly divided into seven groups and harvested at seven different time points: 0 min (control), 30 min (also denoted as 0.5 h), 1 h (hour), 3, 6, 12 and 24 h. For each sample, five seedlings were randomly selected and pooled, with each time point replicated three times. Subsequently, all samples were promptly frozen in liquid nitrogen and stored at −80 °C for subsequent qRT-PCR experiments.
Identification and classification of the TrGRAS genes in white clover
Genomic resources were generously provided by Stig Uggerhøj Andersen from Aarhus University. Arabidopsis GRAS proteins were sourced from the TAIR database [Citation27]. These sequences underwent BLASTP analysis against the white clover genome (all protein sequences) using a set e-value of 1E-05 with an 80% coverage threshold [Citation28]. Hits obtained were then subjected to GRAS domain screening using HMMER software, employing accession number PF03514 and an e-value cutoff of 0.01 [Citation29,Citation30]. Proteins identified as containing the GRAS domain were classified as candidate GRAS proteins; the amino acid sequences of these GRAS proteins are provided in Supplemental Table S1. Comprehensive features, such as gene locus, genome position, protein length and intron numbers, were extracted from the white clover genome. Concurrently, these proteins were categorized into subfamilies based on their homology to Arabidopsis counterparts. They were systematically named TrGRAS001-102 according to their respective subfamily affiliations and genome positions.
Phylogenetic analysis of the TrGRAS genes in white clover
All protein sequences of these TrGRAS genes aligned with each other by using MUSCLE software with default parameters [Citation31]. The MSA (multiple sequence alignment) results were imported into MEGA software [Citation32], and the phylogenetic tree of TrGRAS genes was reconstructed with parameters set as follows: (1) Neighbor-joining (NJ); (2) Poisson correction; (3) bootstrap: 1000 repeat.
Conservative motif analysis of TrGRAS genes in white clover
The conserved motifs of TrGRAS genes were analyzed and identified using MEME software [Citation33], and the MEME analysis was performed with parameters set as (1) the maximum number of motifs: 10; (2) the minimum motif width and maximum motif width: 6 and 50. The search results were visualized using TBtools software [Citation34].
Chromosomal location, gene duplication analysis of GRAS genes in white clover
All white clover proteins were blasted against each other using BLASTP software with e-value set as 0.01, and all blast results were used as input file for MCSanX software to identify genome duplication with default parameters [Citation35]. Then the chromosome distributions and their relationships of TrGRAS genes, including gene position, genome duplication were collected, and these distributions were visualized using CIRCOS software [Citation36].
Gene regulation network analysis of white clover GRAS gene family
The Arabidopsis Gene Regulatory Network (GRN) was acquired from the AraNet database (V2) [Citation37]. To establish homologous gene pairs between white clover and Arabidopsis, a reciprocal BLAST approach was employed. Initially, all white clover proteins were blasted against Arabidopsis proteins with an e-value set to 1E-05, identifying the highest-scoring match as the homologous gene of the respective white clover protein. Subsequently, the same process was performed with Arabidopsis proteins blasted against white clover proteins. Gene pairs between white clover and Arabidopsis proteins were then inferred, facilitating the reconstruction of the white clover GRN based on the Arabidopsis GRN and identified homologous gene pairs. Interactions involving TrGRAS genes were retrieved, and the connections between these genes were extracted. The reconstructed GRN, inclusive of TrGRAS genes, was visualized using Cytoscape software [Citation38]. Functional genes within the GRN were assigned Gene Ontology (GO) annotations, and GO enrichment analysis was conducted using topGO software [Citation39]. This comprehensive approach provides a detailed understanding of the interplay within the gene regulatory network involving TrGRAS genes in white clover.
Expression analysis of TrGRAS genes in response to cold stress
The RNA-seq data were download from NCBI SRA site with accession numbers: PRJNA781064. The transcriptome data was a time-course experiment, including eight stages, the detailed description was listed previously [Citation26]. The high-sequencing reads from this RNA-seq were mapped to white clover genes using Salmon software, and all gene expression levels were estimated and calculated by Salmon’s subroutine quant software [Citation40]. Then the TrGRAS genes expression data were analyzed and clustered using the R program.
qRT-PCR analysis of TrGRAS genes in response to cold stress
Leaves were collected at each time point of stress and ground well with liquid nitrogen. Total RNA extraction from each time point was carried out using the Total Plant RNA Extraction Kit (cat. no. DP315, Tiangen, Beijing, China), with cDNAs subsequently reverse transcribed using the Prime Script RT kit (cat. no. F0922K, Toyobo, Shanghai, China). Primers for two reference genes (actin and GAPDH genes) and TrGRAS genes were designed using Primer3 software [Citation41], and synthesized by Sangon Ltd. Com. (Shanghai, China) (primer details are provided in Supplemental Table S2). qRT-PCR conditions and reaction systems adhered to the SYBR Premix Ex TaqTMII protocol (cat. no. QPK-201, Toyobo, Shanghai, China). qRT-PCR experiments were conducted on a Light Cycler® 96 system (Roche, Rotkreuz, Switzerland), following a program that included initial denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min. Each sample was run with three replicates, and the relative expression of TrGRAS genes was calculated using the 2-ΔΔCt method.
Data analysis
All data analysis was performed with R platform (Version 4.3.1), including statistical analysis and figure plotting.
Results
Identification of GRAS genes in white clover
Through homologous searches and confirmation of the GRAS domain, a total of 102 members were identified in the white clover genome. These GRAS members were designated as TrGRAS001 to TrGRAS102, based on their homology with Arabidopsis and their chromosomal positions (). Analysis of the protein sequences revealed a significant divergence in the size of TrGRAS genes, ranging from 98 amino acids (TrGRAS036) to 1276 amino acids (TrGRAS037), a pattern consistent with GRAS genes observed in alfalfa, another legume forage. Furthermore, a majority of these TrGRAS genes (77 members) were found to lack introns, while twelve members contained two introns, eleven members contained three introns, and only one member (TrGRAS057) contained four introns. These results suggest a high degree of conservation among TrGRAS genes, with indications of potential limited expansion.
Table 1. TrGRAS Genes identified in the white clover genome.
Classification and phylogenetic analysis of TrGRAS genes in white clover
To elucidate the phylogenetic relationships among TrGRAS genes in white clover, an unrooted phylogenetic tree was constructed using all TrGRAS genes (). The analysis revealed the classification of TrGRAS genes into nine distinct subfamilies, consistent with the findings from previous BLAST results. These subfamilies were identified as DELLA (11 members), DLT (two members), HAM (12 members), LISCL (18 members), Os4 (six members), PAT1 (20 members), SCL3 (13 members), SCR (six members) and SHR (14 members). Notably, the LISCL subfamily, comprising 18 members, emerged as one of the larger subfamilies, demonstrating conservatism across various plant species. In contrast, the PAT1 subfamily, with the highest number of members (20), appeared to undergo rapid expansion in the evolutionary process of white clover, a phenomenon not as pronounced in other plant species. These results shed light on the dynamic evolution of the PAT1 subfamily in white clover.
Figure 1. Phylogenetic analysis of the TrGRAS genes in white clover.
Note: The molecular phylogeny of TrGRAS genes in white clover. Red solid circles represent DELLA subfamily; wine red solid triangles represent SCR subfamily; yellow hollow rectangles represent HAM subfamily; blue solid rectangles represent LISCL subfamily; aurantiacus solid diamonds represent SHR subfamily; pink solid triangles represent PAT1 subfamily; green hollow circles represent DLT subfamily; blue hollow rectangles represent SCL3 subfamily; and cyan hollow rectangles represent Os4 subfamily.
Conservative motif analysis of TrGRAS genes in white clover
To further delve into the evolutionary patterns of TrGRAS genes in white clover, a comparative analysis of gene structures was conducted using MEME software (). The results indicated that members within the same subfamilies exhibited similar gene structures, signifying the presence of conserved motifs in GRAS members of the same subfamilies. This observation implies a high degree of conservatism in TrGRAS genes within subfamilies, suggesting potential functional conservation among members of the same subfamilies. Furthermore, the results revealed that motifs within the LISCL subfamily were more conserved compared to the PAT1 subfamily, aligning with our phylogenetic analysis findings. The heightened conservatism in the LISCL subfamily suggests that their functions are likely conserved across different plants. In contrast, the PAT1 subfamily, showing evidence of evolutionary changes, is indicative of more divergent functions among its members.
Figure 2. Distribution of conserved motifs in TrGRAS genes.
Note: The conserved motifs in TrGRAS genes were identified and characterized using MEME software. Each colored box represents the putative motifs detected in the protein sequence.
Chromosome localization, gene duplication analysis of TrGRAS genes in white clover
To elucidate the evolution of TrGRAS genes in white clover, a genome duplication analysis was conducted (). The results revealed an uneven distribution of TrGRAS genes across the white clover genome. Notably, chromosome Tr4O harbored the highest number of TrGRAS genes, with 14 members, while its homologous chromosome Tr4P contained nine TrGRAS genes. This discrepancy suggests divergence between two haplotype genomes during the evolutionary process. Similarly, chromosomes Tr8P and Tr8O exhibited a comparable distribution, but Tr8P contained 12 GRAS members, while Tr8O contained seven. This observation supports the suggestion that two haplotype genomes independently underwent evolution and expansion of TrGRAS genes. Based on BLAST results, a total of 126 gene duplication events were identified, comprising 115 segment duplications (SDs) and 11 tandem duplications (TDs). Genome duplication emerged as the primary force driving the expansion of TrGRAS genes, resulting in the presence of over a hundred GRAS members in white clover. Tandem duplications, on the other hand, played a key role in the divergence between homologous chromosomes. For instance, the presence of two tandem duplication events on chromosome Tr4O, compared to one on Tr4P, contributed to the higher number of TrGRAS members on Tr4O. Similar tandem duplication events were observed on chromosome Tr8P, while Tr8O lacked such events, contributing to the disparity in TrGRAS gene numbers. Despite these duplications leading to the emergence of GRAS clusters on chromosomes, they are considered beneficial for gene expression and functionality.
Figure 3. Chromosomal distribution and gene duplication analysis of the TrGRAS genes in white clover.
Note: All gene duplications were identified using MCSanX software, and they were displayed using CIRCOS software. All TrGRAS genes are labeled with red short line, and genes with duplication event are linked with black curve.
Genetic regulation network analysis of TrGRAS genes in white clover
Gene Regulation Networks (GRN) are widely employed for elucidating the functions of crucial genes in various research studies. In this study, the GRN encompassing TrGRAS genes was reconstructed based on the Arabidopsis interaction database. As depicted in , the GRN comprised 329 genes and 358 interactions. The results highlighted that numerous TrGRAS genes exhibited extensive interactions with other genes. For instance, TrGRAS012 interacted with 68 genes, TrGRAS009 with 57 genes, TrGRAS078 with 34 genes, and TrGRAS052 with 31 genes. This suggests that these TrGRAS genes play pivotal roles in the growth, development and other biological processes in white clover. Subsequently, Gene Ontology (GO) enrichment analysis was conducted on these genes, revealing a focus on regulatory processes (). Enriched terms included ‘regulation of metabolic process’, ‘transcription, DNA-templated’ and ‘gene expression’, among others. Notably, these genes were also enriched in terms such as ‘detection of abiotic stimulus’, ‘detection of light stimulus’ and ‘phosphorelay signal transduction system’, all of which are relevant to temperature stress. This implies the involvement of TrGRAS genes in processes related to temperature stress. Furthermore, the GO analysis results indicated that these genes predominantly localized in the nucleus, aligning with their functions as transcription factors. This observation is further supported by the enrichment in the term ‘transcription regulator activity’.
Figure 4. Gene regulatory network analysis of TrGRAS genes in white clover.
Note: Gene regulatory network of white clover was reconstructed using Arabidopsis gene regulatory network (GRN) from AraNet database. The pink nodes are TrGRAS genes, and the light blue nodes are functional genes interacting withTrGRAS genes in white clover.
Figure 5. GO analysis of gene regulatory networks of TrGRAS genes in white clover.
Note: GO enrichment analysis was performed using topGO package. Red dot BP, green dot MF and blue dot CC represent three types of GO terms, including biological process (BP), molecular function (MF) and cellular component (CC). The dot size represents the number of genes enriched in the GO term. The ordinate is the term of GO, and the abscissa is the p-value of topGO enrichment analysis, −log10 (p).
Expression analysis and qRT-PCR validation of TrGRAS genes in response to cold stress
Previous RNA-seq data was collected to investigate the expression profiles of TrGRAS genes under cold stress, encompassing eight time points from 0 h to 72 h. The results revealed distinct expression profiles of TrGRAS genes in response to cold stress (). All TrGRAS gene expressions were clustered into three groups: Group A, consisting of fifteen TrGRAS genes, exhibited high expression at 1 h under cold stress, indicating rapid responsiveness to cold stress. Group B, comprising twelve TrGRAS genes, demonstrated high expression at 24 h and 72 h under cold stress, suggesting their long-term responsiveness. Group C included 39 TrGRAS genes, with high expression at 6 h and 12 h under cold stress, indicating a moderate response to cold stress. Notably, most genes in Group A belonged to the PAT1 subfamily, such as TrGRAS061, TrGRAS062, TrGRAS063, reinforcing the observation that PAT1 genes exhibit a rapid response to cold stress. Similarly, members of Group B were predominantly from the LISCL subfamily, sharing similar expression profiles. These findings further underscored that genes within the same subfamily exhibit similar expression profiles, indicative of their similar functions.
Figure 6. Expression profile of TrGRAS genes in white clover under cold stress.
Note: The expression levels of TrGRAS genes were collected from the NCBI SRA database (accession number PRJNA781064) and all expression data were clustered and plotted using the function heatmap in R platform.
To validate their expression profiles under cold stress, eight TrGRAS genes were selected for qRT-PCR analysis. The qRT-PCR results confirmed the expression profiles observed in the RNA-seq data (). For instance, TrGRAS038, TrGRAS052, TrGRAS071 and TrGRAS074 exhibited a significant increase under cold stress, aligning with the expressions of TrGRAS genes in Group A. TrGRAS055 displayed the highest expression levels at 6 h and 12 h, suggesting moderate changes in response to cold stress. TrGRAS056, TrGRAS061 and TrGRAS063 demonstrated increased expression at 24 h, implying their prolonged influence in response to cold stress. These results validate the TrGRAS expressions under cold stress, providing valuable insights for exploring their functions in response to cold stress.
Figure 7. qRT-PCR analysis of TrGRAS genes in white clover under cold stress.
Note: The X-axis represents time points in response to cold stress, and the Y-axis represents relative expression levels of TrGRAS genes. The housekeeping genes are Action and GAPDH.
Discussion
White clover is broadly distributed in subtropical and temperate regions, and it has been widely used as a forage crop. It is also used as an important traditional medicine in some countries, such as China, because of abundant bioactive compounds, including phenols, phenolic acids, isoflavones, flavones, flavonols, etc [Citation42]. However, its broad distribution over areas with various environmental conditions have made white clover withstand various abiotic stress, particularly cold stress. Several model plants, including Arabidopsis, rice and soybean, have undergone comprehensive genome sequencing, shedding light on the well-documented GRAS gene families in these models [Citation9,Citation11]. These studies have elucidated the crucial functions of GRAS genes in growth, development and responses to abiotic stress [Citation8]. While the genome of white clover has been sequenced, the exploration of its GRAS genes remained uncharted. In our present research, 102 GRAS genes were identified through homologous searching and domain screening. This number surpassed that of Arabidopsis (32 members) and rice (57 members), aligning closely with soybean (117 members). This observation also correlates with genome duplication events; soybean, being an ancient polyploid, and white clover, an allotetraploid forage with two complete genomes [Citation23]. Consequently, the counts of GRAS genes in soybean and white clover exceeded those in other diploid plants. Moreover, GRAS genes exhibited differentiation between the two progenitor sub-genomes of white clover. For instance, the Tr4O chromosome contained 14 members, while Tr4P held nine TrGRAS genes. Similarly, Tr8P encompassed 12 members, contrasting with Tr8O, which contained seven members. This discrepancy suggests independent evolutionary trajectories for each chromosome, highlighting the dynamic nature of GRAS gene evolution in white clover.
In the expansion of TrGRAS genes, segment duplications (SDs) emerged as a significant driver, contributing to 115 duplication events in white clover. Among these SDs, only eleven were a result of genome duplication, while the remaining 104 events occurred between nonhomologous chromosomes. This suggests a substantial role for nonhomologous SDs in the expansion of GRAS genes during the process of genome enlargement in white clover. Despite the known amplification of SDs during the formation of allotetraploids, these events continued to contribute to the expansion of GRAS genes in white clover. Notably, they enriched the PAT1, SCL3 and SCR subfamilies, leading to functional divergence within TrGRAS genes during the adaptation of white clover to diverse environments [Citation43,Citation44]. Contrastingly, tandem duplications (TDs) occurred less frequently, with only eleven events observed. However, these events played a pivotal role in the chromosomal differentiation of the two diploid progenitor sub-genomes during white clover allopolyploidization. Primarily localized on the Tr4O and Tr8P chromosomes, these TDs contributed to the differentiation of these chromosomes, closely intertwining with gene expression regulation. This mechanism serves as a critical foundation for maintaining genome stability and adapting to diverse environments. The TDs notably expanded the LISCL subfamily, giving rise to four TrGRAS clusters, colloquially referred to as ‘TrGRAS hot harbors’. These clusters exhibited similar expression profiles, exemplified by TrGRAS032, 33 and 34 originating from the same TrGRAS cluster on the Tr8P chromosome, all displaying high expression levels at 24 h and 72 h after cold treatment.
Meanwhile, both RNA-seq data and qRT-PCR analysis have confirmed that a significant proportion of TrGRAS genes respond to cold stress, with the PAT1 subfamily exhibiting the most robust performance. Specifically, members of the PAT1 subfamily exhibit rapid upregulation in response to cold stress, implying their crucial roles in regulating molecular mechanisms involved in cold adaptation. An exemplary case is VaPAT1, which encodes a GRAS transcription factor from grapevine belonging to the PAT1 subfamily. VaPAT1 has been well-demonstrated to regulate jasmonic acid biosynthesis during cold response, enhancing cold, drought and salt tolerance in transgenic plants [Citation17,Citation18]. Within the PAT1 subfamily, eighteen out of the total twenty members were detected as rapidly or intermittently upregulated under cold stress. This suggests that the molecular functions of members in the PAT1 subfamily are divergent, requiring further detailed characterization through molecular experiments. Moreover, the RNA-seq data also revealed the involvement of DELLA, SCR, SCL3 and SHR subfamilies in the regulation of differential responses to cold stress. Members such as TrGRAS001, TrGRAS002, TrGRAS004, TrGRAS008 and TrGRAS010 from the DELLA subfamily exhibited upregulation under cold stress. This subfamily is known to participate in GA signal transduction and may act as downstream factors of the CBF pathway, enhancing cold tolerance by inducing CBF genes [Citation15,Citation16]. Additionally, genes of the SHR subfamily were found to interact with members of the SCR subfamily, contributing to leaf development [Citation44,Citation45]. While many TrGRAS genes primarily function in leaves, it is essential to note that cold stress affects the entire seedling, including root responses. The SHR and SCR genes play a crucial role in regulating cold signals between shoots and roots. Consequently, certain TrGRAS genes were found to exhibit early and rapid upregulation in response to cold stress, while others showed intermittent upregulation across the cold stress process. These findings present potential candidate genes within the TrGRAS family for enhancing cold tolerance in white clover, and their specific functions await determination in future studies.
Conclusions
This research utilized bioinformatics methods, RNA-seq and qRT-PCR experiments to explore GRAS genes in white clover. A total of 102 TrGRAS genes were identified and characterized from whole-genome sequences. Their classification, phylogenetic relationships and conservative motifs were comprehensively examined, revealing nine subfamilies in white clover. Comparative genomics analysis demonstrated that genome duplications played a significant role in the expansion of TrGRAS genes, particularly in the PAT1 and LISCL subfamilies, which were shown to have critical roles in responding to cold stress. Furthermore, RNA-seq and qRT-PCR experiments validated the early or intermittent up-regulation expression profiles of these genes under cold stress, suggesting their potential importance in cold tolerance mechanisms. These findings contribute valuable insights into the molecular regulatory mechanisms of TrGRAS genes in white clover. They hold promise for application in future white clover genetic improvement breeding endeavors.
Authors’ contributions
YS and MH conceived and the study; MH, ML and GZ conducted the experiments; MH, ML, HG and CL analyzed the data; YS and MH wrote the manuscript; MH, ML and YS acquired funding; all authors read and approved the final manuscript.
Supplemental Material
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Data availability statement
The data that support the findings reported in this study are available from the corresponding author [YS] upon reasonable request.
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References
- Singh K, Foley RC, Oñate-Sánchez L. Transcription factors in plant defense and stress responses. Curr Opin Plant Biol. 2002;5(5):430–436. doi: 10.1016/s1369-5266(02)00289-3.
- Chinnusamy V, Schumaker K, Zhu JK. Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants. J Exp Bot. 2004;55(395):225–236. doi: 10.1093/jxb/erh005.
- Markham KK, Greenham K. Abiotic stress through time. New Phytol. 2021;231(1):40–46. doi: 10.1111/nph.17367.
- Zhu J-K. Abiotic stress signaling and responses in plants. Cell. 2016;167(2):313–324. doi: 10.1016/j.cell.2016.08.029.
- Peng J, Carol P, Richards DE, et al. The arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses. Genes Dev. 1997;11(23):3194–3205. doi: 10.1101/gad.11.23.3194.
- Silverstone AL, Ciampaglio CN, Sun T-P The arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway. Plant Cell. 1998;10(2):155–169. doi: 10.1105/tpc.10.2.155.
- Di Laurenzio L, Wysocka-Diller J, Malamy JE, et al. The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the arabidopsis root. Cell. 1996;86(3):423–433. doi: 10.1016/S0092-8674(00)80115-4.
- Jaiswal V, Kakkar M, Kumari P, et al. Multifaceted roles of GRAS transcription factors in growth and stress responses in plants. iScience. 2022;25(9):105026. doi: 10.1016/j.isci.2022.105026.
- Tian C, Wan P, Sun S, et al. Genome-wide analysis of the GRAS gene family in rice and arabidopsis. Plant Mol Biol. 2004;54(4):519–532. doi: 10.1023/B:.PLAN.0000038256.89809.57.
- Grimplet J, Agudelo-Romero P, Teixeira RT, et al. Structural and functional analysis of the GRAS gene family in grapevine indicates a role of GRAS proteins in the control of development and stress responses. Front Plant Sci. 2016;7:353. doi: 10.3389/fpls.2016.00353.
- Wang L, Ding X, Gao Y, et al. Genome-wide identification and characterization of GRAS genes in soybean (Glycine max). BMC Plant Biol. 2020;20(1):415. doi: 10.1186/s12870-020-02636-5.
- Huang W, Xian Z, Kang X, et al. Genome-wide identification, phylogeny and expression analysis of GRAS gene family in tomato. BMC Plant Biol. 2015;15(1):209. doi: 10.1186/s12870-015-0590-6.
- Yang T, Li C, Zhang H, et al. Genome-wide identification and expression analysis of the GRAS transcription in eggplant (solanum melongena L.). Front Genet. 2022;13:932731. doi: 10.3389/fgene.2022.932731.
- Revalska M, Radkova M, Iantcheva A. Functional characterization of Medicago truncatula GRAS7, a member of the GRAS family transcription factors, in response to abiotic stress. Biotechnology & Biotechnological Equipment. 2022;36(1):317–326. doi: 10.1080/13102818.2022.2074893.
- Achard P, Gong F, Cheminant S, et al. The cold-inducible CBF1 factor – dependent signaling pathway modulates the accumulation of the growth-repressing DELLA proteins via its effect on gibberellin metabolism. Plant Cell. 2008;20(8):2117–2129. doi: 10.1105/tpc.108.058941.
- Liu Y, Shi Y, Zhu N, et al. SlGRAS4 mediates a novel regulatory pathway promoting chilling tolerance in tomato. Plant Biotechnol J. 2020;18(7):1620–1633. doi: 10.1111/pbi.13328.
- Yuan Y, Fang L, Karungo SK, et al. Overexpression of VaPAT1, a GRAS transcription factor from vitis amurensis, confers abiotic stress tolerance in arabidopsis. Plant Cell Rep. 2016;35(3):655–666. doi: 10.1007/s00299-015-1910-x.
- Wang Z, Wong DCJ, Wang Y, et al. GRAS-domain transcription factor PAT1 regulates jasmonic acid biosynthesis in grape cold stress response. Plant Physiol. 2021;186(3):1660–1678. doi: 10.1093/plphys/kiab142.
- Xing Z, Huang T, Zhao K, et al. Silencing of sly-miR171d increased the expression of GRAS24 and enhanced postharvest chilling tolerance of tomato fruit. Front Plant Sci. 2022;13:1006940. doi: 10.3389/fpls.2022.1006940.
- Li C, Wang K, Chen S, et al. Genome-wide identification of RsGRAS gene family reveals positive role of RsSHRc gene in chilling stress response in radish (Raphanus sativus L.). Plant Physiol Biochem. 2022;192:285–297. doi: 10.1016/j.plaphy.2022.10.017.
- Wu F, Ma S, Zhou J, et al. Genetic diversity and population structure analysis in a large collection of white clover (Trifolium repens L.) germplasm worldwide. PeerJ. 2021;9:e11325. doi: 10.7717/peerj.11325.
- Inostroza L, Bhakta M, Acuña H, et al. Understanding the complexity of cold tolerance in white clover using temperature gradient locations and a GWAS approach. Plant Genome. 2018;11(3):170096. doi: 10.3835/plantgenome2017.11.0096.
- Griffiths AG, Moraga R, Tausen M, et al. Breaking free: the genomics of allopolyploidy-facilitated niche expansion in white clover. Plant Cell. 2019;31(7):1466–1487. doi: 10.1105/tpc.18.00606.
- Li M, Zhang X, Zhang T, et al. Genome-wide analysis of the WRKY genes and their important roles during cold stress in white clover. PeerJ. 2023;11:e15610. doi: 10.7717/peerj.15610.
- Ma J, Nie G, Yang Z, et al. Genome-wide identification, characterization, and expression profiling analysis of SPL gene family during the inflorescence development in Trifolium repens. Genes. 2022;13(5):900. doi: 10.3390/genes13050900.
- Zhang X, Yang H, Li M, et al. Time-course RNA-seq analysis provides an improved understanding of genetic regulation in response to cold stress from white clover (Trifolium repens L. Biotechnology Biotechnological Equipment. 2022;36(1):745–752.) doi: 10.1080/13102818.2022.2108339.
- Swarbreck D, Wilks C, Lamesch P, et al. The arabidopsis information resource (TAIR): gene structure and function annotation. Nucleic Acids Res. 2008;36(Database issue):D1009–D1014. doi: 10.1093/nar/gkm965.
- Altschul SF, Madden TL, Schäffer AA, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25(17):3389–3402. doi: 10.1093/nar/25.17.3389.
- Finn RD, Clements J, Eddy SR. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 2011;39(Web Server issue):W29–W37. doi: 10.1093/nar/gkr367.
- Punta M, Coggill PC, Eberhardt RY, et al. The pfam protein families database. Nucleic Acids Res. 2011;40(Database issue):D290–D301. doi: 10.1093/nar/gkr1065.
- Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32(5):1792–1797. doi: 10.1093/nar/gkh340.
- Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021;38(7):3022–3027. doi: 10.1093/molbev/msab120.
- Bailey TL, Boden M, Buske FA, et al. MEME suite: tools for motif discovery and searching. Nucleic Acids Res. 2009;37(Web Server issue):W202–W208. doi: 10.1093/nar/gkp335.
- Chen C, Chen H, Zhang Y, et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020;13(8):1194–1202. doi: 10.1016/j.molp.2020.06.009.
- Wang Y, Tang H, DeBarry JD, et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40(7):e49-e49–e49. doi: 10.1093/nar/gkr1293.
- Krzywinski M, Schein J, Birol İ, et al. Circos: an information aesthetic for comparative genomics. Genome Res. 2009;19(9):1639–1645. doi: 10.1101/gr.092759.109.
- Lee T, Yang S, Kim E, et al. AraNet v2: an improved database of co-functional gene networks for the study of Arabidopsis thaliana and 27 other nonmodel plant species. Nucleic Acids Res. 2014;43(Database issue):D996–1002. doi: 10.1093/nar/gku1053.
- Shannon P, Markiel A, Ozier O, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13(11):2498–2504. doi: 10.1101/gr.1239303.
- Alexa A, Rahnenfuhrer J. topGO: enrichment analysis for gene ontology. R package 2019;version 2.38.31.
- Patro R, Duggal G, Love MI, et al. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017;14(4):417–419. doi: 10.1038/nmeth.4197.
- Untergasser A, Cutcutache I, Koressaar T, et al. Primer3 – new capabilities and interfaces. Nucleic Acids Res. 2012;40(15):e115-e115–e115. doi: 10.1093/nar/gks596.
- Ahmad S, Zeb A. Phytochemical profile and pharmacological properties of Trifolium repens. J Basic Clin Physiol Pharmacol. 2021;32(1):20200015. doi: 10.1515/jbcpp-2020-0015.
- Liu Y, Wang W. Characterization of the GRAS gene family reveals their contribution to the high adaptability of wheat. PeerJ. 2021;9:e10811. doi: 10.7717/peerj.10811.
- Lu X, Liu W, Xiang C, et al. Genome-wide characterization of GRAS family and their potential roles in cold tolerance of cucumber (cucumis sativus L.). Int J Mol Sci. 2020;21(11):3857. doi: 10.3390/ijms21113857.
- Gong X, Flores-Vergara MA, Hong JH, et al. SEUSS integrates gibberellin signaling with transcriptional inputs from the SHR-SCR-SCL3 module to regulate Middle cortex formation in the arabidopsis root. Plant Physiol. 2016;170(3):1675–1683. doi: 10.1104/pp.15.01501.