Genome-wide identification and analysis of GRF (growth-regulating factor) gene family in Camila sativa through in silico approaches

2.1 Datasets

The materials for A. thaliana GRF genes were obtained from Plant TFDB (https://planttfdb.gao-lab.org), a plant transcriptional factor database. The various variants, as listed in Supplementary Table 1, were chosen and utilized as a query sequence for C. sativa genes. To evaluate the cis-regulatory elements, the promoter sequence of 1 kilobyte (Kb) upstream of the start codon was downloaded from the Phytozome genome database (https://www.phytozome.net). A BLASTN search was performed from the NCBI database (https://www.ncbi.nlm.nih.gov) to search for candidate genes of C. sativa by using default eating parameters like 10 expectation values, 11 world size, and 25 maximum score numbers in a single line. The cDNAs were retrieved as predicted genes if their e-value meets the set criteria of E ≤ e-10. Further, BLAST searches were also carried out utilizing potential GRF genes as a sequence in the NCBI protein database. The conserved domain was subsequently identified with the use of a local database and the BLASTp program. GRF protein sequences from new plant species that matched C. sativa were identified as GRF genes using the BLASTp program and then mapped on individual chromosomes.

2.2 Gene structure, conserved motifs, and phylogenetic analysis of GRF genes

The TAIR (https://www.arabidopsis.org) database, which is available online, was used for gene analysis. It was used to extract and visualize the organization of exons, introns, and UTRs for individual GRF genes (Rhee et al., 2003). For a schematic depiction of the gene, a gene structure map was created using an online suite called Gene Structure Display Server (http://gsds.gao-lab.org) (Guo et al., 2007). MEME, a sequence analysis program that provides motif-based sequence analysis and is freely available online (https://meme-suite.org), was used to identify protein-conserved motifs (Bailey et al., 2015). The minimum and maximum motif widths were set to 5 and 50, repetition of any number with a distribution of zero or one occurrence per sequence, and all-out motifs were set to 10. To determine evolutionary relationships among GRF genes, the Clustal X version 2.0 (Larkin et al., 2007), with default settings, was used for multiple sequence alignment of the full-length amino acid sequence of GRF proteins from A. thaliana. The neighbor-joining method was adopted with 1000 bootstrap replicates as per the earlier invitation to find relations (Zafar et al., 2020). This method helped construct an unrooted phylogenetic tree using MEGA (molecular evolution genetic analysis) software (Kumar et al., 1994). The evolutionary distances between nine members of the GRF gene family were calculated using this method. The tree was then further presented in circular form among a group of closely related sequences using Interactive Tree Of Life (iTOL) v4 (Letunic and Bork, 2019).

2.3 Gene portrayal and interpretation of GRF structure

The exon-intron structure analysis was performed using the Gene Structure Display Server 2.0 (http://gsds.gao-lab.org/) independent research tool which assists in distinguishing gene structure and comparing it to orthologs in the monocot, dicot, and A. thaliana genomes. The simple modular architecture research tool (SMART) (http://smart.embl-heidelberg.de/) was used to predict and validate the conserved domains based on sequence homology.

2.4 Proteomic analysis of GRF gene family

The protein size, molecular weight (MW), and theoretical isoelectric point (pI) of each GRF protein were measured using the Expy proteomics Server (https://www.expasy.org), proteome database, and sequence analysis tools. Multiple Expectations-Maximization for Motif Elicitation 5.05 (MEME) was utilized to identify protein sequence motifs. The MEME is a collection of numerous ways of discovering and scanning motifs. The MEME motifs found were then looked for using the ScanProsite server (https://prosite.expasy.org/scanprosite/) in the ExPASyProsite database (https://www.expasy.org).

2.5 Protein-protein and DNA-protein interaction and subcellular localization of GRF protein

Protein-protein interaction and subcellular localization of a few GRF proteins from large datasets of GRF proteins were performed by using the String database (https://string-db.org). GRF's DNA-protein interaction was performed by using the accessible online HDOCK web server (http://hdock.phys.hust.edu.cn). A subcellular position of the GRF1 protein was discovered by using the CELLO2GO tool (http://cello.life.nctu.edu.tw/cello2go/).

2.6 Chromosome mapping of GRF genes

The positions of the individual tri-helix genes were retrieved from the corresponding database. All nine GRF genes were shown and mapped to their chromosomal locations on five A. thaliana chromosomes using the TAIR open-access database's chromosome map tool (https://www.arabidopsis.org/jsp/ChromosomeMap/tool.jsp).

2.7 Synteny analysis

To explore the sequence similarity patterns, Circoletto (https://tools.bat.infspire.org/circoletto/) was used to perform synteny analysis and visualize sequence identity among the GRF family genes (Darzentas, 2010).

2.8 Promoter analysis

We retrieved a 1 Kb nucleotide sequence upstream to the start codon for all nine genes using the Phytozome database (http://www.phytozome.net/). These were then subjected to the PLACE cis-regulatory elements database for the identification of already experimentally defined motifs (Higo et al., 1999). Five cis-regulatory elements were obtained, they were CACTFTPPCA1, CURECORECR, GATABOX, ARR1AT, and DOFCOREZM, and their conserved sequences were YCAP, GTAC, GATA, NGATT, and AAAG, respectively. Their locations were then mapped manually.

3.1 Identification and characterization of GRF genes in C. sativa

By using available genomic resources of A. thaliana (Supplementary Table 1), a total of 19 GRF genes includes GRF1(XM_010473979.2), GRF1A (XM_010430916.2), GRF2 (XM_010448327.2), GRF2A (XM_010433733.2), GRF3 (XM_010518559.1), GRF3A (XM_010511173.2), GRF3B ( XM_010506884.2), GRF4 (XM_010517467.2), GRF4A* (XM_010505740.2), GRF4B* (XM_010505738.1), GRF-5(XM_010488877.2), GRF5A (XM_010466969.2), GRF5B (XM_010503161.2), GRF6 (XM_010468817.2), GRF6A (XM_010414605.2), GRF7 (XM_010444638.2), GRF7A (XM_010447680.2), GRF7B (XM_010484472.2), GRF8 (XM_010440777.2), GRF8A (XM_010438107.2), GRF8B (XM_010450302.2), GRF9A* (XM_010519863.2), GRF9B* (XM_010519864.2) and 4 GRF variants GRF4A* (XM_010505740.2), GRF4B* (XM_010505738.1), GRF9A* (XM_010519863.2), GRF9B* (XM_010519864.2) have been identified in the C. sativa genome. The information about their corresponding genomic sequence, coding sequence, and several exons is summarized in Table 1. GRF1, GRF2, GRF2A, and GRF8 had identical and maximal coding areas, i.e. 2046 bp, which encodes 555 amino acids, followed by GRF2. Variants of GRF4 like (GRF4A*, GRF4B*, GRF9A* and GRF9B*) have five similar exons, GRF1, GRF1A, GRF2, GRF2A, GRF3, GRF3A, GRF3B, GRF4, GRF-5, GRF5A, GRF5B, GRF8, GRF8A, GRF8B also has four same exons on each gene, GRF6, GRF6A, GRF7, GRF7A, GRF7B have the same number of exons, i.e., 3. Genes GRF5B and GRF6A were located on identical chromosomes, i.e., 1, GRF3B, GRF4A*, GRF4B* i.e. 4, GRF3, GRF4, GRF9A* and GRF9B* i.e. 6, GRF7 and GRF8 i.e. 11 and GRF2 and GRF8B i.e. 12, GRF5A and GRF6, i.e., 15. The remaining genes (GRF1, GRF1A, GRF2A, GRF3A, GRF5, GRF7A, and GRF7B) were located on 16, 9, 10, 5, 19, 2, and at 18 individually.

The number of introns/exons in C. sativa's 19 GRF genes varies, indicating that GRF genes differ amongst different types of plants. Nevertheless, the most closely related GRF genes, either in their exon lengths or intron numbers, displayed identical exon–intron arrangement and motif composition in the same subfamily. In addition, based on the MEME study, various conserved protein motifs were found in individual GRF proteins (Cao et al., 2016b). The variations among the sub-families in these characteristics showed that the members of the GRF were functionally diversified. Interestingly, all known GRF proteins have motif one and motif two that encode a conserved WRC domain. As seen in Fig. 1, in WRC motifs, zinc-finger structures are closely related, suggesting that this domain functions in DNA binding. The maximum length of the GRF gene in the present study is 2046 bp, with an open reading frame (ORF) encoding 555 amino acids as related to an earlier study of Gnetum luofuense (Hou et al., 2021). The total size of GRF1 in A. thaliana is 1583 bp, which encodes 552 amine acids (Liu et al., 2009). The GRF gene family has been examined in a range of plant species, but no plant orthologs for C. sativa's GRF, GRF4B*, GRF9A*, GRF9B*, or GRF10 genes have been observed. The variability of both exon-intron architectures and motif components of GRF genes could explain the functional differences of GRFs among the plant species investigated.

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Fig. 1

Schematic diagram of the conserved QLQ and WRC domains of all members of GRF gene family.

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3.2 Comparative genome studies of GRF proteins

In the present study, by searching local genome databases, 1 to 9 GRF genes were identified in C. sativa, Oryza sativa, Malus domestica, Zea mays, Carica papaya, Arachis hypogaea, Glycine max, Rosa chinensis, Helianthus annuus, Brassica rapa, Ananas comosua, Capsella rubella, Cicer arietinum, Populus trichocarpa, Ipomoea triloba, Nicotiana tabacum, Spinacia oleracea, Brassica oleracea, Solanum tuberosum, and Coffea arabica. The comparative analysis of GRF genes of C. sativa with other plant species is summarized in Supplementary Table 2. According to comparative genome studies, while the chromosome numbers and genome sizes of different plant species varied, gene ordering within related species remained remarkably conserved across millions of years of evolution (Hardigan et al., 2020; Van de Velde et al., 2016). GRF gene family GRF1 to GRF10 has been already reported in various plant species (Cao et al., 2016a; Ma et al., 2017; Wang et al., 2014). In C. sativa, variants of GRF4 like (GRF4A*, GRF4B*, GRF9A*, and GRF9B*) have five similar exons; however, in the case of Chinese pear (Pyrus bretschneider), poplar (Populous), grape (Vitis vinifera), A. thaliana and rice (Oryza sativa) these all have same 5 number of exons (Cao et al., 2016). The GRF1, GRF1A, GRF2, GRF2A, GRF3, GRF3A, GRF3B, GRF4, GRF-5, GRF5A, GRF5B, GRF8, GRF8A, GRF8B also has four same exons on each gene, in the similar range as the four exons noted for all other plant species (Fonini et al., 2020; Ma et al., 2017; Shang et al., 2018). GRF6, GRF6A, GRF7, GRF7A, and GRF7B have three exons per the exact identification of earlier researchers (Cao et al., 2007; Ma et al., 2017). GRF2 genes encode 555 amino acids with four exons in their gene in the present work. The earlier researcher (Kim, 2019) reported plant-specific duplication of GRF, i.e., GRF2a and GRF2b, which have 555 and 553 amino acids with four exons.

3.3 Physiochemical properties of GRF proteins

To understand the biological functioning of GRF protein, we investigated their characteristics, including the molecular mass, theoretical pI, potential N-glycosylation sites, and functional domains. The molecular weight of GRF proteins ranged from 29.57 to 61.57 kDa (Table 2). The theoretical pI of most GRF proteins ranged between 7.0 and 9.42. In the C. sativa GRF proteins, the number of possible N-glycosylation sites ranged from one to six (Table 2). Figs. 1 and 2 show how the functional domains and motifs of studied GRF proteins were predicted using their protein sequences. As shown in Fig. 1, all GRF proteins have QLQ and WRC domains. Another feature of this domain is the absolute conservation of bulky aromatics or hydrophobic and acidic amino acid residues or their equivalents in chemical and radial properties. The Pro's residue is also fully conserved.

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Fig. 2

Motifs of GRF Genes: Motifs are indicated with conserved regions of amino acid and their different colors code their range.

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For the QLQ domain's role, these amino acid residues, are essential probably for protein–protein interaction. The WRC domain has two peculiar structural properties: many essential amino acids (Arg and Lys) and the motif of C3H, which is the preserved spacing of three Cys and one residue of His and can mediate DNA binding (Zhang and Ghosh, 2001). The essential amino acids are highly conserved, suggesting that they are necessary for the WRC domain function, perhaps as a nuclear localization signal. The present finding is consistent with previous studies indicating that GRF's QLQ and WRC domains are strongly preserved (Cao et al., 2016b; Kim et al., 2003; Wu et al., 2014; Zan et al., 2020).

3.4 Protein-protein and DNA-Protein interaction and subcellular localization of GRF proteins

The network of all GRF families was downloaded from the String database. The networks were divided into clusters of different colors, as shown in Fig. 3. Having 37 nodes, the number of edges is 131, the average number of nodes is 7.08, the average local cluster coefficient is 0, and the expected number of rims is 11. To study in a better way, the interaction network was divided into 9 clusters by using the K-mean cluster. The cluster was composed of closely connected protein interactions, and the balls provided unspecified effects in the interaction network. The arrows show a positive action effect and the one that shows adverse effects. Proteins like the GRF family, Putative leucine-rich repeat receptor-like serine/threonine-protein kinase At2g14440, BEL1-like homeodomain protein five, etc. are presented in Supplementary Fig. 1. The localization probability revealed that GRF1 is mostly localized at the cell's nuclear region with a score of 3.889. As given in Fig. 4, GRF is involved in 44.7% biological functions, 50% molecular functions, and 24.6% cellular functions.

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Fig. 3

All GRF proteins interaction (Protein-protein interaction) with other proteins.

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Fig. 4

A localization probability of GRF1 in Camelina sativa.

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Protein-protein interaction has shown that GRF interacts with other transcription factors such as GRF family proteins, At2g14440 serine/threonine-protein kinase, BEL1-like homeodomain protein 5, etc. These two proteins are directly or indirectly involved in plant reactions to abiotic stresses, including salt stress (Hewezi et al., 2012; Kim et al., 2012). The presence of a QLQ domain for protein–protein interactions, a WRC domain for DNA binding, and a potential nuclear localization signal characterize these proteins. All GRF modules, from 1 to 10, play an essential role in angiosperm growth and development. It determines the ultimate size and form of the leaf organ by regulating the meristematic potential of primordial cells during leaf development. The GRF pair is a pre-requisite for the production of floral organs. In mature leaves, it is also involved in controlling leaf survival and photosynthetic quality. Significantly, the monocot GRF duo also promoted the yield characteristics that guarantee crop productivity, such as grain size and panicle architecture. The GRF gene has a charophyte origin study of GRFs in the most primitive land plants, and charophytes might give information on their significance in an essential lineage of life's evolutionary developmental history (Kim, 2019).

3.5 Phylogenetic analysis of GRF genes

Phylogenetic research was used to accurately explain the evolutionary history of the GRF gene family in plant species. The GRF genes were grouped into five classes according to phylogenetic tree topology. As illustrated in Fig. 5, a phylogenetic examination of C. sativa with other species revealed that all of Camelina sativa's GRF genes were clustered with their homologs. It means that the GRF gene family as a whole is well conserved. GRF genes from C. sativa and other plants have been divided into clades. C. sativa, shown in Fig. 5, is related to Capcella rubella and A. thaliana (Cao et al., 2016). Orthologous pairs of pears and grape GRF proteins were more common, demonstrating that some ancestor GRF genes existed before pear and grape divergence occurred during evolution.

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Fig. 5

Phylogenetic tree analysis of GRFs from Camelina sativa with other species. The various colors represent the different GRF genes.

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3.6 Chromosomal mapping of GRF genes

With the use of data from the public database NCBI, all GRF genes were physically mapped on the chromosomes of O. sativa (GCA_001433935.1) and Z. mays (GCA_902167145.1). C. sativa's (GCA_000633955.1) chromosome 6 has the highest number of GRF genes (3) of all the chromosomes, while the remaining linking chromosome has one of each GRF gene. The pattern of GRF gene distribution on chromosomes also identified distinct physical locations with a higher concentration of GRF genes (Supplementary Fig. 2). The chromosomal analysis found that all GRF genes present in various linkage groups with a maximum linkage group 3 were present in the current sample. The mapping of 9 C. sativa chromosome GRF genes with two other species of O. sativa and Z. mays at chromosome 4, is equivalent to a recent study (FİLİZ et al., 2014). A related GRF gene cluster type ranging from 1 to 9 was found in C. sativa.

3.7 Synteny analysis

The synteny analysis was done using Circoletto. It performed a local alignment and provided a circular output with colorful arcs (Fig. 6). Different colors represent the additional extension of similarity; blue shows the lowest similarity. The increasing likeness to a growing bit score of C. sativa with A. thaliana is shown in green, orange, and red. The majority of genes are orthologs; AT2G22840 and AT4G37740 are orthologs, while CS2G36400 and AT5G53660 are orthologs due to gene duplication. Microsynteny has been found in the studied twenty monocot and dicot plant genomes.

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Fig. 6

The synteny relationship among all the 9 genes of the GRF family generated using Circoletto. The variation of colors represent the extent of similarity and homology among the genes of Camelina sativa. The red color represents the maximum matching portion among the GRF family of Arabidopsis thaliana with Camelina sativa GRF genes.

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3.8 Promoter analysis

Cis-acting regulatory DNA elements are promoter sequence control and expression regulatory elements. On a 1 Kb promoter sequence, five elements were chosen and mapped upstream of the start codons. CACTFTPPCA1, CURECORECR, GATABOX, ARR1AT, and DOFCOREZM were identified as cis-regulatory elements, and their conserved sequences were YCAP, GTAC, GATA, NGATT, and AAAG, respectively. CURECORECR is absent in Cs2G45480 and Cs3G13960, and this cis-regulatory element is present in the smallest proportion in all CsGRF genes. DOFCOREZM is found in large amounts in all CsGRF genes (Supplementary Table 3).