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Functional conservation and divergence in plant-specific GRF gene family revealed by sequences and expression analysis

  • Lingyan Meng , Xiaomeng Li , Yue Hou , Yaxuan Li and Yingkao Hu EMAIL logo
Published/Copyright: March 11, 2022
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Open Life Sciences
From the journal Open Life Sciences Volume 17 Issue 1

Abstract

Unique to plants, growth regulatory factors (GRFs) play important roles in plant growth and reproduction. This study investigated the evolutionary and functional characteristics associated with plant growth. Using genome-wide analysis of 15 plant species, 173 members of the GRF family were identified and phylogenetically categorized into six groups. All members contained WRC and QLQ conserved domains, and the family’s expansion largely depended on segmental duplication. The promoter region of the GRF gene family mainly contained four types of cis-acting elements (light-responsive elements, development-related elements, hormone-responsive elements, and environmental stress-related elements) that are mainly related to gene expression levels. Functional divergence analysis revealed that changes in amino acid site evolution rate played a major role in the differentiation of the GRF gene family, with ten significant sites identified. Six significant sites were identified for positive selection. Moreover, the four groups of coevolutionary sites identified may play a key role in regulating the transcriptional activation of the GRF protein. Expression profiles revealed that GRF genes were generally highly expressed in young plant tissues and had tissue or organ expression specificity, demonstrating their functional conservation with distinct divergence. The results of these sequence and expression analyses are expected to provide molecular evolutionary and functional references for the plant GRF gene family.

Keywords: growth regulatory factors; phylogenetic analysis; positive selection; functional divergence; expression profile

1 Introduction

Transcription factors (TFs), also known as trans-acting factors, bind to DNA in a sequence-specific manner and regulate transcription. They are the main regulators of gene expression and play an important role in plant growth and development, response and adaptation to various stresses, and defense responses [1,2]. Growth regulatory factors (GRFs) are plant-specific TFs that were first discovered in rice (Oryza sativa L.) intercalary meristems named OsGRF1 that play a regulatory role in gibberellic acid (GA)-induced stem extension [3,4]. Subsequently, GRF members were continuously discovered in other plant species, such as Arabidopsis thaliana [5], corn [6], cabbage [7], tomato [8], and wheat [9].

GRF proteins contain conserved WRC (Trp, Arg, and Cys) and QLQ (Gln, Leu, and Gln) domains at the N-terminal [4,7,10,11]. The WRC domain contains a nuclear localization signal and a C3H zinc finger structure, similar to the plant-specific Cys–Cys–Cys–His (CX9CX10CX2H) motif, and participates in DNA binding [3,12,13]. The QLQ domain is composed of a highly conserved Gln–Leu–Gln (QX3LX2Q) residue and neighboring residues that are similar to the N-terminal of the chromatin remodeling complex (SWI2/SNF2) in yeast and can interact with SNF11 [14]. In addition, the lengths and amino acid sequences of the C-terminal of GRF proteins are different, but they still have the common characteristics of TFs [11]. Studies have shown that the lack of complete C-terminal will lead to the loss of transcriptional activation activity, which will affect plant growth [6], and there are TQL, GGPL, and FFD domains at the C-terminus of partial plants. The QLQ domain also participates in the interaction with the SYT N-terminal homology (SNH) domain at the N-terminal of the GRF-interacting factor (GIF) protein to form a transcriptional activator [15]. As a transcriptional coactivator, GIF promotes gene alteration of the GRF protein. GRF and GIF genes are highly expressed in almost all meristems [16,17] and participate in regulating the growth and development of some tissues and organs in plants; interestingly, GRF/GIF-mediated growth is usually associated with enhanced cell proliferation or cell expansion [15,18]. Previous studies have shown that microRNA396 (miR396) is involved in the regulation of GRF gene expression. MiR396 regulates most GRF members after transcription and fine-tunes their expression to control the GRF/GIF-dependent process [19,20]. Kim [16] showed that miR396 could regulate the proliferation of cells and the size of meristems.

In the model plant Arabidopsis, AtGRF1, 2, and 3 control the size of plant leaves by reorganizing the size of plant cells. For instance, compared with the wild type, overexpression of AtGRF1 and AtGRF2 leads to larger leaves and cotyledons or leads to a late flowering phenotype [11,21]. GRF4 is not only involved in the expansion and growth of leaf cells but is also necessary for the development of cotyledons and shoot apex meristems [22]. Importantly, a study demonstrated that GRF5 could promote the duration of the cell proliferation phase during leaf development [23]. Similarly, GIF1 coordinates cell proliferation in different cell layers in rice by translocating through plasmodesmata [16]. Moreover, GRF participates in the osmotic resistance of plants, and AtGRF7 mutants are more resistant to drought and salt stress than wild type and AtGRF7 overexpression lines [13]. Additionally, the regulation mechanism of GRF expression is complex. The miR396/GRF regulatory network has an adjusted effect on flower organ development [24]. In tomatoes, miR396 has two mature types (miR396a and miR396b). The study by Cao et al. found that downregulation of miR396a and miR396b results in an overall upregulation of target GRFs, resulting in the obvious enlargement of flowers, sepals, and fruits [8]. Furthermore, miR396–GRF/GIF has been important in regulating plant senescence and affects different stages of leaf development. It can coordinate plant growth and physiological responses with endogenous and environmental signals [18]. AtGRF3 lines with mutations in miR396 binding site and lines overexpressing AtGRF5 have delayed leaf senescence [25]. In Arabidopsis, ectopic overexpression of miR396 inhibits the expression of GRF genes and inhibits the transcriptional coactivator GIF1. The transcriptional expression of GRFs was also regulated by GA3, which enhances the expression of OsGRF1, 2, 3, 7, 8, 10, and 12 [10]. Studies have shown that GRF protein can also affect plant growth and development by negatively regulating the expression of dehydration-response element-binding (DREB2A) protein and knotted-like homeobox protein [26,27]. GRFs also participate in plant ear development [28], root growth [29,30], floral organogenesis [31], apical meristem growth and maintenance [26,32], and other processes.

To better understand the dynamics of the evolution of the GRF gene and the functional relationship between gene family members, we constructed a phylogenetic tree of 15 plants species. Additionally, we conducted an analysis of gene duplication methods once tandem and segment duplication events may lead to the generation of new gene family members. Of note, we also looked for positive selection sites and coevolution sites, usually related to protein functions in the protein evolution process. The expression profile obtained through the transcriptome data reflects the conservation and difference of GRF genes in different tissues. The organizational differences in the cis-acting regulatory elements in the promoter region can partially explain the differences in their expression.

2 Materials and methods

2.1 Identification of GRF gene family

The A. thaliana GRF gene sequences were downloaded from the TAIR database (http://www.arabidopsis.org/) and used as seed sequences to identify GRF gene family members in 15 sequenced plant species (Brachypodium distachyon, O. sativa, Sorghum bicolor, Zea mays, Physcomitrella patens, Selaginella moellendorffii, A. thaliana, Brassica rapa, Citrus sinensis, Glycine max, Gossypium raimondii, Medicago sativa, Phaseolus vulgaris, Populus trichocarpa, and Solanum lycopersicum) using the BLASTP tool in the Phytozome database (http://www.phytozome.org). These 15 species represent the plant kingdom from lower to higher plants. An E-value ≤ 1 × 10−5 and a complete open reading frame were used as the selection criteria for protein sequences. Pfam (http://pfam.xfam.org) and SMART (http://smart.embl-heidelberg.de/) proteomics service programs were used to verify whether the candidate GRF protein contains conserved WRC(PF08879) and QLQ(PF08880) domains. In addition, the protein sequences, coding sequences (CDS), genomic sequences, and 2,000 bp sequences upstream of the start codon were obtained from the Phytozome database. The amino acid number, isoelectric point (pI), and molecular weight (MW) of GRF proteins were obtained from the ExPASy database (https://www.expasy.org/) [33].

2.2 Phylogenetic tree construction and analysis of exon–intron structure, motif, and cis-acting elements

Multiple sequence alignment of all amino acid sequences of the identified full-length GRF proteins was conducted using the MUSCLE program [34,35]. Based on the multiple sequence alignment files, MEGA7.0 was used to build phylogenetic trees under the default parameters using neighbor-joining (NJ) and maximum likelihood (ML) methods, with bootstrap set to 1,000 [36]. In addition, MrBayes 3.2.5 software was used to build a Bayesian evolutionary tree [37].

The exon–intron structure of GRFs was derived from the online tool GSDS (http://gsds.cbi.pku.edu.cn/) by comparing the CDS and genomic sequences [38]. Conserved motifs were detected using the MEME program (http://meme-suite.org/tools/meme) [39], which was run with the maximum number of motifs set to 20, whereas the remaining parameters were preset by the system.

The 2,000-bp upstream sequence of the start codon was collected from the Phytozome database (www.phytozome.net), and cis-acting elements of known sequences were analyzed using the PlantCARE online service platform (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [40]. Five members (orange1.1g047108m, orange1.1g028751m, orange1.1g007514m, Brara.I03590, Solyc10g083510) with incomplete promoter sequences were manually deleted to exclude them from subsequent analyses.

2.3 Duplication event analysis

The synonymous substitution rates (K s) of GRF gene pairs produced by duplication events were identified using the Plant Genome Duplication Database (http://chibba.agtec.uga.edu/duplication), and segmental duplicated gene pairs with K s values greater than one and anchor loci less than three were excluded [41,42]. The K-estimator method was used to calculate the K s, K a, and K a/K s [43]. The approximate date of segmental duplication events was estimated using T = K s/2λ formula. According to earlier researches, the λ value of the approximate date used for the calculation of the duplication events are as follows: 1.5 × 10−8 for Arabidopsis [5], 6.5 × 10−9 for B. distachyon [44], 1.4 × 10−8 for B. rapa [45], 6.1 × 10−9 for G. max [44], 6.5 × 10−9 for O. sativa [46], 9.1 × 10−7 for P. trichocarpa [47], 1.5 × 10−8 for G. raimondii [48], and 6.5 × 10−9 for Z. mays [49]. The Phytozome database (http://www.phytozome.org) was used to identify tandem duplication gene pairs. On the same chromosome, if the TF family members are contained within ten genes before and after a homologous gene, it is proved that these two homologous genes resulted from tandem duplication [41].

2.4 Functional divergence, positive selection, and coevolution analyses

The DIVERGE software (version 3.0) was used to detect the Type I and Type II functional divergence sites in different groups of the GRF gene family through posterior analysis [50]. The Type I and Type II functional divergence coefficients (θ I and θ II) between members of a subfamily were obtained to measure the degree of divergence, such that if θ I or θ II were significantly greater than 0, then it can be said that certain amino acid sites underwent significant changes in their evolution rates or physiochemical properties, respectively [51,52]. The Q k value is an important indicator for measuring the degree of functional divergence at the amino acid site and is directly proportional to the probability of any functional divergence between two subfamilies [52]. In this study, the critical value of Q k was set to 0.8.

Positive selection was investigated using a maximum-likelihood approach using site models and branch site models in the CODEML program of PAML v4.4 [53,54]. Comparing the two models, null models (M0 and M7) and alternative hypothesis models (M3 and M8) were executed for positive selection identification. A likelihood ratio test (LRT) was performed according to the chi-square distribution, and then the alternative hypothesis model was established based on the p value. The Bayes empirical Bayes (BEB) method was used to calculate the posterior probabilities [55].

The coevolution amino acid site was calculated by coevolution analysis using protein sequences with the PERL software. BLOSUM-corrected amino acid distances were used to identify amino acid covariations [56].

2.5 Protein structure prediction

Construction of the 3D structure of GRF proteins was done using the online website PHYRE2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) [57,58], and important amino acid sites on the 3D structure were marked using the PyMOL v1.7.4 software (Schrödinger, Inc.).

2.6 Expression analysis of GRFs

RNA-seq data of four different plant species were obtained from the following websites: Arabidopsis eFP Browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) [59], soybean eFP Browser (http://bar.utoronto.ca/efpsoybean/cgi-bin/efpWeb.cgi) [60], rice eFP Browser (http://www.bar.utoronto.ca/efprice/cgi-bin/efpWeb.cgi) [61], and Brachypodium eFP Browser (http://bar.utoronto.ca/efp_brachypodium/cgi-bin/efpWeb.cgi). Then, the GenePattern (http://www.broadinstitute.org/cancer/software/genepattern/) software tool was used to construct the expression heat map.

3 Results

3.1 Genome-wide identification of GRF gene family members

Sequences of nine GRF gene family members in the Arabidopsis genome were obtained from the TAIR database and used as seed sequences. Using the BLASTP tool in the Phytozome database, 173 members of the GRF gene family were identified in 15 species. In addition, results show that the values of pIs ranged from 4.78 to 10.57 (Table S1), with an average value of 8.12, and that 79.7% of GRF protein members were weakly alkaline, whereas only 20.3% were weakly acidic. The number of amino acid residues was between 155 and 817, with an average of 406, and the MW was between 22.555 and 86.039 kDa. These data show that to adapt to changes in the external environment and meet the functional requirements of different periods in the long-term evolution process, members of the GRF gene family underwent changes in their respective physical and chemical properties.

3.2 Phylogenetic relationships and molecular characterization of GRF gene family

Multiple sequence alignment of the 173 proteins identified was performed, and a NJ phylogenetic tree (Figure A1), maximum-likelihood phylogenetic tree (Figure A2), and a Bayesian phylogenetic tree (Figure 1a) were constructed. Comparative analysis showed that the three trees had similar topological structures, proving the reliability of phylogenetic tree results, and the Bayesian tree was used for subsequent analysis. Phylogenetic analysis divided the GRFs into six subfamilies, namely Groups I, II, III, IV, V, and VI, containing 36, 23, 18, 41, 22, and 33 members, respectively. Moreover, phylogenetic tree analysis showed that Groups I, II, IV, and VI contained both monocots and eudicots (Figure 1a), whereas Group III only contains eudicots. Except for one member of Group V is Selaginella, all the others are eudicots. Pteridophyta and bryophyta are mainly distributed in Group IV. Based on the structural characteristics of the phylogenetic tree and the analysis of subfamily members, it can be deduced that all family members have a common ancestor. During the evolution process, the GRF family members, including lower plants, differentiated first and evolved into the Group IV. The differentiation of Group III and V was thought to have occurred after the monocots and eudicots split. The evolutionary pattern of GRF TFs was significantly different between monocots and eudicots.

Figure 1 Phylogenetic relationships and exon–intron structures of GRF gene family members: (a) Bayesian phylogenetic tree of the GRF gene family. The color of subclades indicates the six corresponding gene subfamilies. Blue, black, red, yellow, green, and purple represent Groups I, II, III, IV, V, and VI, respectively. Gene names of different species are represented by different colors. (b) Exon–intron structures of the GRF genes. Yellow bars: exons; lines: introns; blue bars: 3′ untranslated region. (c) Distributions of conserved motifs. Twenty putative motifs are indicated in different colored boxes.
Figure 1

Phylogenetic relationships and exon–intron structures of GRF gene family members: (a) Bayesian phylogenetic tree of the GRF gene family. The color of subclades indicates the six corresponding gene subfamilies. Blue, black, red, yellow, green, and purple represent Groups I, II, III, IV, V, and VI, respectively. Gene names of different species are represented by different colors. (b) Exon–intron structures of the GRF genes. Yellow bars: exons; lines: introns; blue bars: 3′ untranslated region. (c) Distributions of conserved motifs. Twenty putative motifs are indicated in different colored boxes.

Figure 2 Schematic of 3D structure of plant GRF protein: (a) 3D structure of AT3G13960. The QLQ and WRC domains are colored in green and magenta, respectively. Yellow indicates Type I functional divergence sites and orange indicates positive selection sites. (b) The sites responsible for the coevolution sites are colored in red.
Figure 2

Schematic of 3D structure of plant GRF protein: (a) 3D structure of AT3G13960. The QLQ and WRC domains are colored in green and magenta, respectively. Yellow indicates Type I functional divergence sites and orange indicates positive selection sites. (b) The sites responsible for the coevolution sites are colored in red.

Figure 3 Expression profiles of (a) A. thaliana and (b) B. distachyon GRF genes. Reddest (hot) and greenest (cool) shades denote higher and lower expression levels, respectively. Gray indicates the value of 0 in the original RNA sequence data, and the software automatically recognizes this part of the data as “missing.”
Figure 3

Expression profiles of (a) A. thaliana and (b) B. distachyon GRF genes. Reddest (hot) and greenest (cool) shades denote higher and lower expression levels, respectively. Gray indicates the value of 0 in the original RNA sequence data, and the software automatically recognizes this part of the data as “missing.”

The results of exon–intron analysis (Figure 1b) showed that most members of Groups I, II, and VI were composed of three exons, and other members contained two or four exons. In Groups III, IV, and V, most members contained four exons, and a few members, such as X417311, X412762, and AT4G24150, contained six exons. Another member, Solyc08g068760, contained only one exon. The results of exon–intron further prove the reliability of the phylogenetic tree branch. Differences in the number of exons may have been due to the loss or acquisition of exons during long-term evolution. Furthermore, the number of exons in higher plants decreased, which may be due to the loss of introns.

The potential motif structures in the GRF family were obtained, and 20 conserved motifs, designated as motifs 1–20, were identified (Figure 1c). Members of the same subfamily contained a similar number and sequence of motif types. All members had motifs 1 and 2, which contained the WRC and QLQ domains, respectively. In addition, the highest numbers and types of motifs were observed in Group IV, including the bryophyta, pteridophyte, monocots, and eudicots. It is worth noting that different subfamilies contained unique motifs; Group I contained motifs 7, 10, and 14; Group III contained motif 15; moreover, motifs 19 and 20 were present in Group IV, and motif 11 was specific to Group VI. This may be related to the functional divergence among various subfamilies during evolution, supporting the classification.

Figure 4 Expression profiles of (a) O. sativa and (b) G. max GRF genes. Reddest (hot) and greenest (cool) shades denote higher and lower expression levels, respectively. Gray indicates the value of 0 in the original RNA sequence data, and the software automatically recognizes this part of the data as “missing.”
Figure 4

Expression profiles of (a) O. sativa and (b) G. max GRF genes. Reddest (hot) and greenest (cool) shades denote higher and lower expression levels, respectively. Gray indicates the value of 0 in the original RNA sequence data, and the software automatically recognizes this part of the data as “missing.”

3.3 Analysis of cis-acting elements in the promoter region of GRF genes

To better understand the expression and function of the GRFs, seven types of cis-acting elements in the promoter region of the GRF gene family were identified, namely light-responsive elements, development-related elements, hormone-responsive elements, environmental stress-related elements, site-binding related elements, promoter-related elements, and other elements. Among them, light-responsive elements, development-related elements, hormone-responsive elements, and environmental stress-related elements were closely related to plant growth regulation.

We analyzed light-responsive cis-acting elements, such as Box4, GT1-motif, G-box, ATCT-motif, and TCT-motif, which was the most abundant type in the GRF gene family (Table S2). Box4, G-box, GT1-motif, and TCT-motif were abundant, with average copies of 1.74, 1.12, 0.91, and 0.66, respectively. For example, G-box has widely existed in the promoters of light-controlled genes and other environmental factors regulating genes. It has a highly conserved core sequence CACGTG, a universal regulatory element for plants responding to external environmental stimuli. Hormone responsive elements, including the GARE-motif, TCA-element, CGTCA-motif, TGACG-motif, and abscisic acid-responsive element (ABRE), which are involved in response to gibberellin, auxin, methyl jasmonic acid, and abscisic acid, have also been identified. Of these, the most abundant was ABRE, and the average copies of ABRE, ERE, CTCCA-motif, and TGACG-motif were 1.52, 1.29, 0.95, and 0.94, respectively. In addition, CCGTCC-box (12.37%), CAT-box (14.68%), and O2-site (12.58%) were identified as the development-related elements; CCGTCC-box participated in the development of meristems. Meanwhile, we identified the cis-acting elements in response to environmental stress: LTR, MBS, WUN-motif, GC-motif, ARE, and TC-rich repeats. The WUN-motif, TC-rich repeats, and LTR were damage response elements, stress response elements, and low-temperature response elements, respectively, indicating that the GRF genes were related to defense recovery and temperature change response. The existence of these elements indicates that a variety of environmental factors may regulate GRF gene expression.

In addition, the composition of cis-acting elements between different subfamilies is not only similar, but also differentiated. Several types of cis-acting elements have a large distribution among each subfamily, such as G-box, ARE, as-1, ERE, and ABRE, and they have different functions. ARE is an essential cis-acting regulatory element involved in anaerobic induction. The difference in cis-acting elements in different subfamilies also supported the differentiation of GRF genes between different subfamilies, thus promoting functional divergence during evolution.

3.4 Gene duplication event of GRFs

Gene duplication is one of the main mechanisms for the establishment of new gene functions and biological evolution. It is well known that gene duplication can occur in multiple ways, including segmental duplication, tandem duplication, and transposition events, which provide raw materials for evolutionary mechanisms [62]. In this study, we mainly focused on segmental and tandem duplication. Tandem duplication events usually produce multiple family members within the same or adjacent intergenic regions [63]. We only found two pairs of tandem duplication genes from soybeans (Glyma.17G232700 and Glyma.17G232600; and Glyma.U028700 and Glyma.U028600), which were members of Group IV. This showed that tandem duplication accounted for a small proportion of the evolution of the GRF gene.

In addition, 40.5% of the GRF gene family members were associated with segmental duplication events (Table S3). Segmental duplication events were most active in eudicots. Among Arabidopsis, cabbage, soybean, cotton, and poplar, a total of 51 genes were confirmed to be segment duplicated genes. Segmented duplication events of eudicots were distributed in all groups, whereas segmented duplication events in monocotyledonous plants were mainly concentrated in Groups, I II, and VI. Considering together, our results suggest that segmental duplication promoted the expansion of the GRF gene family. Interestingly, segmental and tandem duplication events were both found in Group IV, suggesting that both types of duplications contributed to the expansion of Group IV. In parallel, both genes in each duplicate gene pair belonged to the same group. These genes might be not undergoing functional divergence during the evolution process.

Large-scale duplication events generate a large number of homologous genes. Table S3 lists the average K s values (synonymous base substitution rates) and estimated dates for the segmental duplication events of the GRF gene family in eight plant species. As shown in the table, most of the segment duplication gene pairs should have been generated and retained along with whole-genome duplications (WGDs), such as Arabidopsis, G. raimondii, O. sativa, and B. rapa. In some species, such as P. trichocarpa and Z. mays, partial segment duplication events are earlier than WGDs event. It is speculated that the remaining segment duplication genes may have originated from independent repeat events. In the process of gene replication, mutations may occur, leading to functional divergence and species diversification among the GRF gene subfamily members.

3.5 Functional divergence in the GRF gene family

To further analyze whether amino acid substitutions in the GRF gene family lead to functional divergence, the Type I functional divergence coefficient (θ I) between two groups was detected by posterior analysis using DIVERGEv3.0 [50]. Results ranged from 0.023 to 0.689 and were significantly greater than 0 (Table 1). Except for Groups II and III, and II and IV, the LRT values among other groups reached extremely significant levels (p < 0.01), which indicated that plant GRF protein had sites that underwent Type I functional divergence during the evolutionary process. A similar analysis showed no Type II functional divergence sites related to the physicochemical properties of amino acid residues. The degree of type II functional divergence (θ II) is not significantly greater than 0, and the detected sites are not statistically significant.

Table 1

Functional divergence sites among groups of the GRF gene family

Group 1 Group 2 Type I Type II
θ I ± s.e. LRT Q k > 0.8 θ II ± s.e. Q k > 0.8
I II 0.265 ± 0.071 5.394* 41V −0.155 ± 0.202 None
I III 0.337 ± 0.125 4.200* 39V −0.058 ± 0.153 None
I IV 0.188 ± 0.075 1.246* None −0.096 ± 0.153 None
I V 0.427 ± 0.082 16.799** 27H,30L,46I,9K,88R −0.020 ± 0.183 None
I VI 0.181 ± 0.183 0.408 None −0.045 ± 0.130 None
II III 0.023 ± 0.022 0 None −0.262 ± 0.228 None
II IV 0.289 ± 0.098 6.100* 41V −0.120 ± 0.210 None
II V 0.159 ± 0.107 0.013 None −0.163 ± 0.254 None
II VI 0.030 ± 0.022 0 None −0.247 ± 0.204 None
III IV 0.036 ± 0.113 7.255* 39V,109E −0.031 ± 0.158 27H,113H
III V 0.369 ± 0.088 4.291* 39V,109E −0.038 ± 0.188 27H
III VI 0.182 ± 0.220 2.418* None 0.009 ± 0.135 None
IV V 0.057 ± 0.100 1.244* None −0.038 ± 0.194 None
IV VI 0.635 ± 0.192 6.270* 16P,39V −0.047 ± 0.134 113H
V VI 0.689 ± 0.171 7.919* 27H,46I,79K 0.022 ± 0.171 None

Note: θ I and θ II: the coefficients of Type-I and Type-II functional divergence. LRT: likelihood ratio test. *: p < 0.05, **: p < 0.01, highly significant. Q k : posterior probability.

By calculating the posterior probability of each site (Q k > 0.8), the key amino acid sites related to functional divergence of the GRF gene were determined. The results revealed 10 Type I functional divergence sites (9G, 16P, 27H, 30L, 39V, 41V, 46I, 79K, 88R, and 109E). Of these sites, simultaneous changes in the evolution rate and physicochemical properties of site 27H were observed. Our results suggest that the functional difference between any two groups was mainly due to the difference in their amino acid evolutionary rates.

3.6 Positive selection, coevolution, and three-dimensional structural analysis of the GRF gene family

Site-specific models for positive selection were used to determine sites that underwent positive selection during evolution. By comparing M0 (one ratio) and M3 (discrete) models, we calculated the twice log-likelihood difference of the models and obtained 2ΔlnL = 932.8146 (p < 0.01, df = 4). This LRT result was statistically significant, indicating that the amino acids in the GRF gene family have experienced variable selection pressures among sites. Comparing the M7 (beta) and M8 models (beta and ω), the 2ΔlnL was 5795.66, and the ω value implanted in the M8 model was 2.55821, which was much greater than 1 (Table 2). Then, the BEB was used to evaluate the posterior probability of the location considered to be a positive choice. A total of six positively selective sites were detected in the M8 model, and their posterior probabilities were all greater than 0.95. Of these sites, one (36V; p < 0.05) was at a significant level, and five (19P, 20T, 39V, 80P, 84P; p < 0.01) were at an extremely significant level (Table 2).

Table 2

Positive selection test of plant GRFs using site-specific models

Model InL a 2ΔlnL Estimate of parameters Positively selected sitesb
M0 −12532.1472 932.8146** ω = 0.08401 Not allowed
M3 −12065.7399 p0 = 0.36332, p1 = 0.42999, p2 = 0.20668, ω0 = 0.00414, ω1 = 0.08165, ω2 = 0.28340 None
M7 −12032.7693 5299.5134** p = 0.41819, q = 3.82194 Not allowed
M8 −14682.5260 p0 = 0.99999, p = 0.71594, q = 1.59678, p1 = 0.00001, ω = 2.55821 19P**, 20T**, 36V*, 39V**, 80P**, 84P**

Note: a: log likelihood. b: Positive selection sites are inferred at posterior probabilities >95%. *: p < 0.05, **: p < 0.01.

We identified four groups of coevolution sites in the GRF gene family: 215H and 216A; 217S and 272S; 395T and 396G; and 252R, 266S, 391S, 390H, and 389S. These sites were distributed at the C-terminus with transcriptional activity and were not far apart in the tertiary structure, indicating that their compensatory mutations contributed to local stability maintenance.

The GRF gene family member AT3G13960 from Arabidopsis was used as a representative to construct the 3D structure. As shown in Figure 2a, the identified positive selection sites marked on the 3D structure were mostly distributed on the QLQ and WRC domains, and three sites were placed on the alpha helix. These results indicate that the positive selection sites have undergone adaptive evolution in the process of evolution and may also play an important role in maintaining the stability of the protein structure. Similarly, the Type I functional divergence sites were also mainly distributed in the QLQ and WRC domains (Figure 2a). We speculate that these sites may be involved in protein–protein interactions. The detected coevolution sites were mainly distributed at the C-terminus of the GRF protein (Figure 2b), and they are related to each other during the evolution process as one of the sites cannot be mutated alone. The C-terminus of GRF protein has transcriptional activity, suggesting that these amino acid sites may play a vital role in maintaining protein transcriptional activity, which reflects the conservation of protein structure and function.

3.7 Expression profiling of GRF genes

To analyze the function of the GRF gene and explore its expression in different organs and developmental stages, we selected four species (A. thaliana, O. sativa, G. max, and B. distachyon) for expression profile analysis using published RNA-seq data. All members of the GRF gene family in rice (Figure 4a) have the characteristic upregulated expression in the stem apex meristem (SAM), as well as in soybean GRF gene family members (Figure 4b), indicating that GRF gene family members have conservative characteristics and functions related to growth and development in SAM. In addition, different subfamily members from the same species have different expression profiles in different developmental tissues. For example, the GRF gene family members from Arabidopsis (Figure 3a) have different expression profiles in flowers, leaves, shoots, and seeds, and with especially high expression in shoots and seeds. Moreover, the expression profiles at different developmental stages of the same tissue were slightly different, and expression level in the early stages was higher than that in the later stages. The GRF gene family members LOC_Os11g35030 and Glyma.17G232600 from Group IV both exhibited high expression characteristics in SAM, and both contained cis-regulatory elements called as-1 that are involved in root-specific expression, and are closely related to root growth and development. Analysis of the expression profiles of the four plants showed that the expression of GRF gene members in different subfamilies was similar and differentiated among members.

4 Discussion

4.1 Molecular characterization and genomic analysis of the GRF gene family

Genome-wide analysis of 15 species identified 173 TF family members, and the GRF gene family was divided into six groups, namely Groups I–VI, by phylogenetic analysis (Figure 1a). The distribution of each species was different; only P. trichocarpa, G. raimondii, and S. lycopersicum were common in the six groups. This is different from the five subfamily classifications of Cao et al. [64], probably, because there are more species used in our research included lower and higher plants represented all of the plant kingdom. However, the number of introns and exons in each group was similar, suggesting that GRF structure was conserved within a branch. The GRF gene-coding region of eudicots was mainly composed of four exons, whereas that of monocotyledonous plants was mainly composed of three exons (Figure 1b). Moreover, results showed that 20 motifs were detected, with at least one or two conserved motif types and spatial arrangement patterns in the same subfamily. All GRFs contained motifs 1 and 2 (Figure A2), which meant that the WRC and QLQ conserved domains at the N-terminal region were present in all screened GRF proteins. The GRF protein interacts with the GIF protein through the QLQ domain, forming a functional complex with a transcriptional activation function. Earlier studies have shown that in Arabidopsis, compared with other AtGRF family members, AtGRF5 (AT3G13960) binds to AtGIF more tightly and plays a greater role in cell proliferation in the leaf primordia [65]. In brief, there are obvious differences between each group, and the functions of GRF members within the same subfamily have certain similarities. These special domains may specifically combine with other molecules to function.

Gene duplication events or the formation of new species can lead to diversification of protein functions. The functional difference between duplication genes is caused by the accumulation of repeated mutations at amino acid sites [66]. According to our results, 45 pairs of segmental duplication genes were detected in eight species (Table S3), and only two pairs of tandem duplication genes were found in soybean, indicating that the amplification of the GRF gene family mainly relied on segmental duplication, and these segmental duplication genes can be retained in different species through whole genome duplication events, which was consistent with the study of Chen et al. in soybeans [67].

In addition, according to the research of Fonini et al., it is speculated that the earliest duplication event of GRF gene was the replication event in the common ancestor of charophyte and land plants [68].

By analyzing the functional divergence of the GRF gene family, ten Type I functional divergence sites were detected in this experiment, indicating that changes in the evolution rate of amino acid sites was the main driving force for the functional differentiation of the GRF gene family. Phylogenetic analysis has confirmed that positive selection contributes to protein evolution, and that changes in positive selection sites allow proteins to acquire new catalytic functions without injuring their main biochemical properties [69]. Six sites of positive selective (19P, 20T, 36V, 39V, 80P, 84P; Table 2) in the GRF gene family were revealed, and most of these sites were located in the conserved domains (Figure 2a), suggesting that they may play an indispensable role in the interaction with the (SNH) domains of the plant GIF protein. We speculate whether the changes in these positive selection sites may interfere with the formation of complexes between GRF and GIF proteins, thus affecting the activation of GRF protein, and the growth and shape of leaves and petals [15].

It was found that there was an amino acid site (39V) that experienced both Type I functional divergence and positive selection. The change in the evolutionary rate of this amino acid site was also favorable for alleles to improve fitness, and this may be one of the important evolutionary forces for functional divergence after gene duplication. It is believed that this amino acid site may have a substantial role in maintaining the stability of the GRF protein.

The presence of intramolecular coevolutionary networks is also one of the factors that determine the evolution of proteins. The complexity of evolution is related to the potential functional and structural interactions between sites [70]. Four groups of coevolution sites were found to be located at the C-terminus of the GRF protein (Figure 2b). The C-terminal domain is more functionally diverse compared to the conserved N-terminal domain [11]. Wu et al. found that although ZmGRF10 contains a complete N-terminal domain, its transcriptional activation activity is lost due to the lack of a complete C-terminal domain, thus affecting plant growth [6]. We suspect that these coevolution sites at the C-terminal domain will affect the transcriptional activity of the GRF protein, which is worthy of further investigation. These coevolution sites reflect the conservation of protein functions or structures.

4.2 Expression and potential functions of GRFs

According to the analysis of phylogenetic trees obtained from multiple species, GRF family members with high homology and similar structures usually clustered together. Therefore, the function of known GRF gene family members can be used to predict the function of unknown members in the same branch. Tissue-specific expression profiles may also be similar among different species in the same subfamily. Among the members of GRFs expressed in flower organs and meristems, AtGRF7, 8, and 9 (AT5G53660, AT4G24150, AT2G45480) are involved in pistil development, and AtGRF8 is particularly important in the late development of floral organs [13,21,24]. According to the expression profiles obtained, it can be hypothesized that the soybean family members Glyma06G134600 and Glyma04G230600 that belonged to Group V with ATGRF7 and 8, were also highly expressed in flowers (Figures 3a and 4b). We believe that they also played a role in the development of soybean flower organs. Studies have shown that the expression of AtGRF7 is related to the regulatory mechanism of abscisic acid-responsive elements (ABRE). AREBs/ABFs can activate DREB2A transcription through ABRE in response to osmotic stress, whereas AtGRF7 can bind to the promoter region of DREB2A to inhibit osmotic stress or abscisic acid response and prevent growth inhibition [13]. Soybean GRFs that are part of Group V also contain ABRE cis-acting elements, which are speculated to be related to osmotic stress and abscisic acid response [71]. In addition, the research on abiotic stress of soybean found that the morphology of soybean under shading conditions has undergone great changes, including reduced leaf area and weight, and excessive elongation of stems [67]. As mentioned earlier, many light-responsive regulatory elements have been detected in the GRF gene. We speculate that shaded conditions affect the expression of light-responsive elements in GRF gene and have an impact on the growth and development of soybeans.

Based on the expression profiles, it was found that most GRF members in rice are usually highly expressed in SAM and young inflorescences. OsGRF1, which was preferentially expressed in the stem apex containing the SAM and the younger leaf primordia, can regulate the growth of leaves. Another member of Group I, LOC_Os06g10310, showed a trend of upregulation in SAM (Figure 4a). Similarly, in soybeans, different GRF subfamily members were highly expressed in the meristems, and analysis of cis-acting elements showed that most members contained cis-acting regulatory elements related to meristem expression and meristem specific activation, such as CAT-box and CCGTCC-box1. At the same time, according to the research on other plants, the expression of GRF gene in wheat shoot tip meristem is significantly higher than that in other tissues [72], In tobacco, it is found that NtabGRF gene is highly expressed in active growing tissues and responds to various hormone treatments [73]. All results confirmed that the GRF gene is highly expressed in the vigorously divided tissues of plants, and it is speculated that the GRF gene plays an important role in the early stage of plant growth and development. We hypothesize that the expression similarity of genes in different tissues of the same species is greater than their expression similarity in the same tissue of different species.

However, it can be seen that Arabidopsis GRFs in different subfamilies also have different expression profiles in the same tissues. AtGRF3 and 1, and AtGRF2 from Groups III and IV, respectively, were highly expressed in the roots, whereas AtGRF7 and 8 from Group V were relatively downregulated in roots. This may have been caused by functional divergence during evolution. In addition, GRF regulates root growth through MIR396a, which affects the extension zone and regulates root growth. It was also found that B. distachyon GRFs from Groups I and VI (Bradi3g57267, Bradi1g46427, Bradi5g20670, Bradi1g50597, and Bradi3g52547) were highly expressed in young leaves, whereas the members of Groups I and VI in soybean showed a trend of decreased expression in young leaves; they also did not have similar expression profiles in similar tissues (Figures 3b and 4b). This may also indicate functional divergence caused by gene duplication during the evolutionary process.

5 Conclusion

Genome-wide analysis of 15 plant species identified 173 members of the GRF gene family, which were divided into six subfamilies. The molecular structure characteristics, phylogeny, gene duplication, and expression patterns in different tissue analyses revealed an evolutionarily conserved transcriptional activity of the GRF gene family. Type-I functional divergence was identified as the main reason for the functional diversification of GRFs, and positive selection sites played an important role in domain differentiation. The appearance of multiple cis-acting elements indicated that GRFs were regulated by diverse hormones and environmental factors. Members of the same subfamily contained similar cis-acting elements, and their expression profiles reflected the conservation of GRF gene family members; however, the differences of GRFs between species also reflected the differentiation of GRF gene family members during evolution. This study provides useful information for further exploration of the molecular evolution mechanism and functional features of the plant GRF gene family.

  1. Funding information: This study was supported by the Natural Science Foundation of Beijing, China (6192002).

  2. Conflict of interest: Authors state no conflict of interest.

  3. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Appendix

Figure A1 NJ phylogenetic tree of all the GRF gene family. Blue, black, red, yellow, green, and purple represent Groups I, II, III, IV, V, and VI, respectively.
Figure A1

NJ phylogenetic tree of all the GRF gene family. Blue, black, red, yellow, green, and purple represent Groups I, II, III, IV, V, and VI, respectively.

Figure A2 ML phylogenetic tree of all the GRF gene family. Blue, black, red, yellow, green, and purple represent Groups I, II, III, IV, V, and VI, respectively.
Figure A2

ML phylogenetic tree of all the GRF gene family. Blue, black, red, yellow, green, and purple represent Groups I, II, III, IV, V, and VI, respectively.

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Received: 2021-04-23
Revised: 2021-12-03
Accepted: 2022-01-03
Published Online: 2022-03-11

© 2022 Lingyan Meng et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

  1. Biomedical Sciences
  2. Effects of direct oral anticoagulants dabigatran and rivaroxaban on the blood coagulation function in rabbits
  3. The mother of all battles: Viruses vs humans. Can humans avoid extinction in 50–100 years?
  4. Knockdown of G1P3 inhibits cell proliferation and enhances the cytotoxicity of dexamethasone in acute lymphoblastic leukemia
  5. LINC00665 regulates hepatocellular carcinoma by modulating mRNA via the m6A enzyme
  6. Association study of CLDN14 variations in patients with kidney stones
  7. Concanavalin A-induced autoimmune hepatitis model in mice: Mechanisms and future outlook
  8. Regulation of miR-30b in cancer development, apoptosis, and drug resistance
  9. Informatic analysis of the pulmonary microecology in non-cystic fibrosis bronchiectasis at three different stages
  10. Swimming attenuates tumor growth in CT-26 tumor-bearing mice and suppresses angiogenesis by mediating the HIF-1α/VEGFA pathway
  11. Characterization of intestinal microbiota and serum metabolites in patients with mild hepatic encephalopathy
  12. Functional conservation and divergence in plant-specific GRF gene family revealed by sequences and expression analysis
  13. Application of the FLP/LoxP-FRT recombination system to switch the eGFP expression in a model prokaryote
  14. Biomedical evaluation of antioxidant properties of lamb meat enriched with iodine and selenium
  15. Intravenous infusion of the exosomes derived from human umbilical cord mesenchymal stem cells enhance neurological recovery after traumatic brain injury via suppressing the NF-κB pathway
  16. Effect of dietary pattern on pregnant women with gestational diabetes mellitus and its clinical significance
  17. Potential regulatory mechanism of TNF-α/TNFR1/ANXA1 in glioma cells and its role in glioma cell proliferation
  18. Effect of the genetic mutant G71R in uridine diphosphate-glucuronosyltransferase 1A1 on the conjugation of bilirubin
  19. Quercetin inhibits cytotoxicity of PC12 cells induced by amyloid-beta 25–35 via stimulating estrogen receptor α, activating ERK1/2, and inhibiting apoptosis
  20. Nutrition intervention in the management of novel coronavirus pneumonia patients
  21. circ-CFH promotes the development of HCC by regulating cell proliferation, apoptosis, migration, invasion, and glycolysis through the miR-377-3p/RNF38 axis
  22. Bmi-1 directly upregulates glucose transporter 1 in human gastric adenocarcinoma
  23. Lacunar infarction aggravates the cognitive deficit in the elderly with white matter lesion
  24. Hydroxysafflor yellow A improved retinopathy via Nrf2/HO-1 pathway in rats
  25. Comparison of axon extension: PTFE versus PLA formed by a 3D printer
  26. Elevated IL-35 level and iTr35 subset increase the bacterial burden and lung lesions in Mycobacterium tuberculosis-infected mice
  27. A case report of CAT gene and HNF1β gene variations in a patient with early-onset diabetes
  28. Study on the mechanism of inhibiting patulin production by fengycin
  29. SOX4 promotes high-glucose-induced inflammation and angiogenesis of retinal endothelial cells by activating NF-κB signaling pathway
  30. Relationship between blood clots and COVID-19 vaccines: A literature review
  31. Analysis of genetic characteristics of 436 children with dysplasia and detailed analysis of rare karyotype
  32. Bioinformatics network analyses of growth differentiation factor 11
  33. NR4A1 inhibits the epithelial–mesenchymal transition of hepatic stellate cells: Involvement of TGF-β–Smad2/3/4–ZEB signaling
  34. Expression of Zeb1 in the differentiation of mouse embryonic stem cell
  35. Study on the genetic damage caused by cadmium sulfide quantum dots in human lymphocytes
  36. Association between single-nucleotide polymorphisms of NKX2.5 and congenital heart disease in Chinese population: A meta-analysis
  37. Assessment of the anesthetic effect of modified pentothal sodium solution on Sprague-Dawley rats
  38. Genetic susceptibility to high myopia in Han Chinese population
  39. Potential biomarkers and molecular mechanisms in preeclampsia progression
  40. Silencing circular RNA-friend leukemia virus integration 1 restrained malignancy of CC cells and oxaliplatin resistance by disturbing dyskeratosis congenita 1
  41. Endostar plus pembrolizumab combined with a platinum-based dual chemotherapy regime for advanced pulmonary large-cell neuroendocrine carcinoma as a first-line treatment: A case report
  42. The significance of PAK4 in signaling and clinicopathology: A review
  43. Sorafenib inhibits ovarian cancer cell proliferation and mobility and induces radiosensitivity by targeting the tumor cell epithelial–mesenchymal transition
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  45. Active legumain promotes invasion and migration of neuroblastoma by regulating epithelial-mesenchymal transition
  46. Effect of cell receptors in the pathogenesis of osteoarthritis: Current insights
  47. MT-12 inhibits the proliferation of bladder cells in vitro and in vivo by enhancing autophagy through mitochondrial dysfunction
  48. Study of hsa_circRNA_000121 and hsa_circRNA_004183 in papillary thyroid microcarcinoma
  49. BuyangHuanwu Decoction attenuates cerebral vasospasm caused by subarachnoid hemorrhage in rats via PI3K/AKT/eNOS axis
  50. Effects of the interaction of Notch and TLR4 pathways on inflammation and heart function in septic heart
  51. Monosodium iodoacetate-induced subchondral bone microstructure and inflammatory changes in an animal model of osteoarthritis
  52. A rare presentation of type II Abernethy malformation and nephrotic syndrome: Case report and review
  53. Rapid death due to pulmonary epithelioid haemangioendothelioma in several weeks: A case report
  54. Hepatoprotective role of peroxisome proliferator-activated receptor-α in non-cancerous hepatic tissues following transcatheter arterial embolization
  55. Correlation between peripheral blood lymphocyte subpopulations and primary systemic lupus erythematosus
  56. A novel SLC8A1-ALK fusion in lung adenocarcinoma confers sensitivity to alectinib: A case report
  57. β-Hydroxybutyrate upregulates FGF21 expression through inhibition of histone deacetylases in hepatocytes
  58. Identification of metabolic genes for the prediction of prognosis and tumor microenvironment infiltration in early-stage non-small cell lung cancer
  59. BTBD10 inhibits glioma tumorigenesis by downregulating cyclin D1 and p-Akt
  60. Mucormycosis co-infection in COVID-19 patients: An update
  61. Metagenomic next-generation sequencing in diagnosing Pneumocystis jirovecii pneumonia: A case report
  62. Long non-coding RNA HOXB-AS1 is a prognostic marker and promotes hepatocellular carcinoma cells’ proliferation and invasion
  63. Preparation and evaluation of LA-PEG-SPION, a targeted MRI contrast agent for liver cancer
  64. Proteomic analysis of the liver regulating lipid metabolism in Chaohu ducks using two-dimensional electrophoresis
  65. Nasopharyngeal tuberculosis: A case report
  66. Characterization and evaluation of anti-Salmonella enteritidis activity of indigenous probiotic lactobacilli in mice
  67. Aberrant pulmonary immune response of obese mice to periodontal infection
  68. Bacteriospermia – A formidable player in male subfertility
  69. In silico and in vivo analysis of TIPE1 expression in diffuse large B cell lymphoma
  70. Effects of KCa channels on biological behavior of trophoblasts
  71. Interleukin-17A influences the vulnerability rather than the size of established atherosclerotic plaques in apolipoprotein E-deficient mice
  72. Multiple organ failure and death caused by Staphylococcus aureus hip infection: A case report
  73. Prognostic signature related to the immune environment of oral squamous cell carcinoma
  74. Primary and metastatic squamous cell carcinoma of the thyroid gland: Two case reports
  75. Neuroprotective effects of crocin and crocin-loaded niosomes against the paraquat-induced oxidative brain damage in rats
  76. Role of MMP-2 and CD147 in kidney fibrosis
  77. Geometric basis of action potential of skeletal muscle cells and neurons
  78. Babesia microti-induced fulminant sepsis in an immunocompromised host: A case report and the case-specific literature review
  79. Role of cerebellar cortex in associative learning and memory in guinea pigs
  80. Application of metagenomic next-generation sequencing technique for diagnosing a specific case of necrotizing meningoencephalitis caused by human herpesvirus 2
  81. Case report: Quadruple primary malignant neoplasms including esophageal, ureteral, and lung in an elderly male
  82. Long non-coding RNA NEAT1 promotes angiogenesis in hepatoma carcinoma via the miR-125a-5p/VEGF pathway
  83. Osteogenic differentiation of periodontal membrane stem cells in inflammatory environments
  84. Knockdown of SHMT2 enhances the sensitivity of gastric cancer cells to radiotherapy through the Wnt/β-catenin pathway
  85. Continuous renal replacement therapy combined with double filtration plasmapheresis in the treatment of severe lupus complicated by serious bacterial infections in children: A case report
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  87. Preclinical immunogenicity assessment of a cell-based inactivated whole-virion H5N1 influenza vaccine
  88. One case of iodine-125 therapy – A new minimally invasive treatment of intrahepatic cholangiocarcinoma
  89. S1P promotes corneal trigeminal neuron differentiation and corneal nerve repair via upregulating nerve growth factor expression in a mouse model
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  91. Calcifying nanoparticles initiate the calcification process of mesenchymal stem cells in vitro through the activation of the TGF-β1/Smad signaling pathway and promote the decay of echinococcosis
  92. Evaluation of prognostic markers in patients infected with SARS-CoV-2
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  94. Characterization of the structural, oxidative, and immunological features of testis tissue from Zucker diabetic fatty rats
  95. Effects of glucose and osmotic pressure on the proliferation and cell cycle of human chorionic trophoblast cells
  96. Investigation of genotype diversity of 7,804 norovirus sequences in humans and animals of China
  97. Characteristics and karyotype analysis of a patient with turner syndrome complicated with multiple-site tumors: A case report
  98. Aggravated renal fibrosis is positively associated with the activation of HMGB1-TLR2/4 signaling in STZ-induced diabetic mice
  99. Distribution characteristics of SARS-CoV-2 IgM/IgG in false-positive results detected by chemiluminescent immunoassay
  100. SRPX2 attenuated oxygen–glucose deprivation and reperfusion-induced injury in cardiomyocytes via alleviating endoplasmic reticulum stress-induced apoptosis through targeting PI3K/Akt/mTOR axis
  101. Aquaporin-8 overexpression is involved in vascular structure and function changes in placentas of gestational diabetes mellitus patients
  102. Relationship between CRP gene polymorphisms and ischemic stroke risk: A systematic review and meta-analysis
  103. Effects of growth hormone on lipid metabolism and sexual development in pubertal obese male rats
  104. Cloning and identification of the CTLA-4IgV gene and functional application of vaccine in Xinjiang sheep
  105. Antitumor activity of RUNX3: Upregulation of E-cadherin and downregulation of the epithelial–mesenchymal transition in clear-cell renal cell carcinoma
  106. PHF8 promotes osteogenic differentiation of BMSCs in old rat with osteoporosis by regulating Wnt/β-catenin pathway
  107. A review of the current state of the computer-aided diagnosis (CAD) systems for breast cancer diagnosis
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  109. A novel association between Bmi-1 protein expression and the SUVmax obtained by 18F-FDG PET/CT in patients with gastric adenocarcinoma
  110. The role of erythrocytes and erythroid progenitor cells in tumors
  111. Relationship between platelet activation markers and spontaneous abortion: A meta-analysis
  112. Abnormal methylation caused by folic acid deficiency in neural tube defects
  113. Silencing TLR4 using an ultrasound-targeted microbubble destruction-based shRNA system reduces ischemia-induced seizures in hyperglycemic rats
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  116. Cloning and prokaryotic expression of WRKY48 from Caragana intermedia
  117. Novel Brassica hybrids with different resistance to Leptosphaeria maculans reveal unbalanced rDNA signal patterns
  118. Application of exogenous auxin and gibberellin regulates the bolting of lettuce (Lactuca sativa L.)
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  155. Development of alkaline phosphatase-scFv and its use for one-step enzyme-linked immunosorbent assay for His-tagged protein detection
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  164. Evolution trend of soil fertility in tobacco-planting area of Chenzhou, Hunan Province, China
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  171. Erratum
  172. Erratum to “Changes of immune cells in patients with hepatocellular carcinoma treated by radiofrequency ablation and hepatectomy, a pilot study”
  173. Erratum to “A two-microRNA signature predicts the progression of male thyroid cancer”
  174. Retraction
  175. Retraction of “Lidocaine has antitumor effect on hepatocellular carcinoma via the circ_DYNC1H1/miR-520a-3p/USP14 axis”
https://doi.org/10.1515/biol-2022-0018
Keywords for this article
growth regulatory factors; phylogenetic analysis; positive selection; functional divergence; expression profile
Creative Commons
BY 4.0

Articles in the same Issue

  1. Biomedical Sciences
  2. Effects of direct oral anticoagulants dabigatran and rivaroxaban on the blood coagulation function in rabbits
  3. The mother of all battles: Viruses vs humans. Can humans avoid extinction in 50–100 years?
  4. Knockdown of G1P3 inhibits cell proliferation and enhances the cytotoxicity of dexamethasone in acute lymphoblastic leukemia
  5. LINC00665 regulates hepatocellular carcinoma by modulating mRNA via the m6A enzyme
  6. Association study of CLDN14 variations in patients with kidney stones
  7. Concanavalin A-induced autoimmune hepatitis model in mice: Mechanisms and future outlook
  8. Regulation of miR-30b in cancer development, apoptosis, and drug resistance
  9. Informatic analysis of the pulmonary microecology in non-cystic fibrosis bronchiectasis at three different stages
  10. Swimming attenuates tumor growth in CT-26 tumor-bearing mice and suppresses angiogenesis by mediating the HIF-1α/VEGFA pathway
  11. Characterization of intestinal microbiota and serum metabolites in patients with mild hepatic encephalopathy
  12. Functional conservation and divergence in plant-specific GRF gene family revealed by sequences and expression analysis
  13. Application of the FLP/LoxP-FRT recombination system to switch the eGFP expression in a model prokaryote
  14. Biomedical evaluation of antioxidant properties of lamb meat enriched with iodine and selenium
  15. Intravenous infusion of the exosomes derived from human umbilical cord mesenchymal stem cells enhance neurological recovery after traumatic brain injury via suppressing the NF-κB pathway
  16. Effect of dietary pattern on pregnant women with gestational diabetes mellitus and its clinical significance
  17. Potential regulatory mechanism of TNF-α/TNFR1/ANXA1 in glioma cells and its role in glioma cell proliferation
  18. Effect of the genetic mutant G71R in uridine diphosphate-glucuronosyltransferase 1A1 on the conjugation of bilirubin
  19. Quercetin inhibits cytotoxicity of PC12 cells induced by amyloid-beta 25–35 via stimulating estrogen receptor α, activating ERK1/2, and inhibiting apoptosis
  20. Nutrition intervention in the management of novel coronavirus pneumonia patients
  21. circ-CFH promotes the development of HCC by regulating cell proliferation, apoptosis, migration, invasion, and glycolysis through the miR-377-3p/RNF38 axis
  22. Bmi-1 directly upregulates glucose transporter 1 in human gastric adenocarcinoma
  23. Lacunar infarction aggravates the cognitive deficit in the elderly with white matter lesion
  24. Hydroxysafflor yellow A improved retinopathy via Nrf2/HO-1 pathway in rats
  25. Comparison of axon extension: PTFE versus PLA formed by a 3D printer
  26. Elevated IL-35 level and iTr35 subset increase the bacterial burden and lung lesions in Mycobacterium tuberculosis-infected mice
  27. A case report of CAT gene and HNF1β gene variations in a patient with early-onset diabetes
  28. Study on the mechanism of inhibiting patulin production by fengycin
  29. SOX4 promotes high-glucose-induced inflammation and angiogenesis of retinal endothelial cells by activating NF-κB signaling pathway
  30. Relationship between blood clots and COVID-19 vaccines: A literature review
  31. Analysis of genetic characteristics of 436 children with dysplasia and detailed analysis of rare karyotype
  32. Bioinformatics network analyses of growth differentiation factor 11
  33. NR4A1 inhibits the epithelial–mesenchymal transition of hepatic stellate cells: Involvement of TGF-β–Smad2/3/4–ZEB signaling
  34. Expression of Zeb1 in the differentiation of mouse embryonic stem cell
  35. Study on the genetic damage caused by cadmium sulfide quantum dots in human lymphocytes
  36. Association between single-nucleotide polymorphisms of NKX2.5 and congenital heart disease in Chinese population: A meta-analysis
  37. Assessment of the anesthetic effect of modified pentothal sodium solution on Sprague-Dawley rats
  38. Genetic susceptibility to high myopia in Han Chinese population
  39. Potential biomarkers and molecular mechanisms in preeclampsia progression
  40. Silencing circular RNA-friend leukemia virus integration 1 restrained malignancy of CC cells and oxaliplatin resistance by disturbing dyskeratosis congenita 1
  41. Endostar plus pembrolizumab combined with a platinum-based dual chemotherapy regime for advanced pulmonary large-cell neuroendocrine carcinoma as a first-line treatment: A case report
  42. The significance of PAK4 in signaling and clinicopathology: A review
  43. Sorafenib inhibits ovarian cancer cell proliferation and mobility and induces radiosensitivity by targeting the tumor cell epithelial–mesenchymal transition
  44. Characterization of rabbit polyclonal antibody against camel recombinant nanobodies
  45. Active legumain promotes invasion and migration of neuroblastoma by regulating epithelial-mesenchymal transition
  46. Effect of cell receptors in the pathogenesis of osteoarthritis: Current insights
  47. MT-12 inhibits the proliferation of bladder cells in vitro and in vivo by enhancing autophagy through mitochondrial dysfunction
  48. Study of hsa_circRNA_000121 and hsa_circRNA_004183 in papillary thyroid microcarcinoma
  49. BuyangHuanwu Decoction attenuates cerebral vasospasm caused by subarachnoid hemorrhage in rats via PI3K/AKT/eNOS axis
  50. Effects of the interaction of Notch and TLR4 pathways on inflammation and heart function in septic heart
  51. Monosodium iodoacetate-induced subchondral bone microstructure and inflammatory changes in an animal model of osteoarthritis
  52. A rare presentation of type II Abernethy malformation and nephrotic syndrome: Case report and review
  53. Rapid death due to pulmonary epithelioid haemangioendothelioma in several weeks: A case report
  54. Hepatoprotective role of peroxisome proliferator-activated receptor-α in non-cancerous hepatic tissues following transcatheter arterial embolization
  55. Correlation between peripheral blood lymphocyte subpopulations and primary systemic lupus erythematosus
  56. A novel SLC8A1-ALK fusion in lung adenocarcinoma confers sensitivity to alectinib: A case report
  57. β-Hydroxybutyrate upregulates FGF21 expression through inhibition of histone deacetylases in hepatocytes
  58. Identification of metabolic genes for the prediction of prognosis and tumor microenvironment infiltration in early-stage non-small cell lung cancer
  59. BTBD10 inhibits glioma tumorigenesis by downregulating cyclin D1 and p-Akt
  60. Mucormycosis co-infection in COVID-19 patients: An update
  61. Metagenomic next-generation sequencing in diagnosing Pneumocystis jirovecii pneumonia: A case report
  62. Long non-coding RNA HOXB-AS1 is a prognostic marker and promotes hepatocellular carcinoma cells’ proliferation and invasion
  63. Preparation and evaluation of LA-PEG-SPION, a targeted MRI contrast agent for liver cancer
  64. Proteomic analysis of the liver regulating lipid metabolism in Chaohu ducks using two-dimensional electrophoresis
  65. Nasopharyngeal tuberculosis: A case report
  66. Characterization and evaluation of anti-Salmonella enteritidis activity of indigenous probiotic lactobacilli in mice
  67. Aberrant pulmonary immune response of obese mice to periodontal infection
  68. Bacteriospermia – A formidable player in male subfertility
  69. In silico and in vivo analysis of TIPE1 expression in diffuse large B cell lymphoma
  70. Effects of KCa channels on biological behavior of trophoblasts
  71. Interleukin-17A influences the vulnerability rather than the size of established atherosclerotic plaques in apolipoprotein E-deficient mice
  72. Multiple organ failure and death caused by Staphylococcus aureus hip infection: A case report
  73. Prognostic signature related to the immune environment of oral squamous cell carcinoma
  74. Primary and metastatic squamous cell carcinoma of the thyroid gland: Two case reports
  75. Neuroprotective effects of crocin and crocin-loaded niosomes against the paraquat-induced oxidative brain damage in rats
  76. Role of MMP-2 and CD147 in kidney fibrosis
  77. Geometric basis of action potential of skeletal muscle cells and neurons
  78. Babesia microti-induced fulminant sepsis in an immunocompromised host: A case report and the case-specific literature review
  79. Role of cerebellar cortex in associative learning and memory in guinea pigs
  80. Application of metagenomic next-generation sequencing technique for diagnosing a specific case of necrotizing meningoencephalitis caused by human herpesvirus 2
  81. Case report: Quadruple primary malignant neoplasms including esophageal, ureteral, and lung in an elderly male
  82. Long non-coding RNA NEAT1 promotes angiogenesis in hepatoma carcinoma via the miR-125a-5p/VEGF pathway
  83. Osteogenic differentiation of periodontal membrane stem cells in inflammatory environments
  84. Knockdown of SHMT2 enhances the sensitivity of gastric cancer cells to radiotherapy through the Wnt/β-catenin pathway
  85. Continuous renal replacement therapy combined with double filtration plasmapheresis in the treatment of severe lupus complicated by serious bacterial infections in children: A case report
  86. Simultaneous triple primary malignancies, including bladder cancer, lymphoma, and lung cancer, in an elderly male: A case report
  87. Preclinical immunogenicity assessment of a cell-based inactivated whole-virion H5N1 influenza vaccine
  88. One case of iodine-125 therapy – A new minimally invasive treatment of intrahepatic cholangiocarcinoma
  89. S1P promotes corneal trigeminal neuron differentiation and corneal nerve repair via upregulating nerve growth factor expression in a mouse model
  90. Early cancer detection by a targeted methylation assay of circulating tumor DNA in plasma
  91. Calcifying nanoparticles initiate the calcification process of mesenchymal stem cells in vitro through the activation of the TGF-β1/Smad signaling pathway and promote the decay of echinococcosis
  92. Evaluation of prognostic markers in patients infected with SARS-CoV-2
  93. N6-Methyladenosine-related alternative splicing events play a role in bladder cancer
  94. Characterization of the structural, oxidative, and immunological features of testis tissue from Zucker diabetic fatty rats
  95. Effects of glucose and osmotic pressure on the proliferation and cell cycle of human chorionic trophoblast cells
  96. Investigation of genotype diversity of 7,804 norovirus sequences in humans and animals of China
  97. Characteristics and karyotype analysis of a patient with turner syndrome complicated with multiple-site tumors: A case report
  98. Aggravated renal fibrosis is positively associated with the activation of HMGB1-TLR2/4 signaling in STZ-induced diabetic mice
  99. Distribution characteristics of SARS-CoV-2 IgM/IgG in false-positive results detected by chemiluminescent immunoassay
  100. SRPX2 attenuated oxygen–glucose deprivation and reperfusion-induced injury in cardiomyocytes via alleviating endoplasmic reticulum stress-induced apoptosis through targeting PI3K/Akt/mTOR axis
  101. Aquaporin-8 overexpression is involved in vascular structure and function changes in placentas of gestational diabetes mellitus patients
  102. Relationship between CRP gene polymorphisms and ischemic stroke risk: A systematic review and meta-analysis
  103. Effects of growth hormone on lipid metabolism and sexual development in pubertal obese male rats
  104. Cloning and identification of the CTLA-4IgV gene and functional application of vaccine in Xinjiang sheep
  105. Antitumor activity of RUNX3: Upregulation of E-cadherin and downregulation of the epithelial–mesenchymal transition in clear-cell renal cell carcinoma
  106. PHF8 promotes osteogenic differentiation of BMSCs in old rat with osteoporosis by regulating Wnt/β-catenin pathway
  107. A review of the current state of the computer-aided diagnosis (CAD) systems for breast cancer diagnosis
  108. Bilateral dacryoadenitis in adult-onset Still’s disease: A case report
  109. A novel association between Bmi-1 protein expression and the SUVmax obtained by 18F-FDG PET/CT in patients with gastric adenocarcinoma
  110. The role of erythrocytes and erythroid progenitor cells in tumors
  111. Relationship between platelet activation markers and spontaneous abortion: A meta-analysis
  112. Abnormal methylation caused by folic acid deficiency in neural tube defects
  113. Silencing TLR4 using an ultrasound-targeted microbubble destruction-based shRNA system reduces ischemia-induced seizures in hyperglycemic rats
  114. Plant Sciences
  115. Seasonal succession of bacterial communities in cultured Caulerpa lentillifera detected by high-throughput sequencing
  116. Cloning and prokaryotic expression of WRKY48 from Caragana intermedia
  117. Novel Brassica hybrids with different resistance to Leptosphaeria maculans reveal unbalanced rDNA signal patterns
  118. Application of exogenous auxin and gibberellin regulates the bolting of lettuce (Lactuca sativa L.)
  119. Phytoremediation of pollutants from wastewater: A concise review
  120. Genome-wide identification and characterization of NBS-encoding genes in the sweet potato wild ancestor Ipomoea trifida (H.B.K.)
  121. Alleviative effects of magnetic Fe3O4 nanoparticles on the physiological toxicity of 3-nitrophenol to rice (Oryza sativa L.) seedlings
  122. Selection and functional identification of Dof genes expressed in response to nitrogen in Populus simonii × Populus nigra
  123. Study on pecan seed germination influenced by seed endocarp
  124. Identification of active compounds in Ophiopogonis Radix from different geographical origins by UPLC-Q/TOF-MS combined with GC-MS approaches
  125. The entire chloroplast genome sequence of Asparagus cochinchinensis and genetic comparison to Asparagus species
  126. Genome-wide identification of MAPK family genes and their response to abiotic stresses in tea plant (Camellia sinensis)
  127. Selection and validation of reference genes for RT-qPCR analysis of different organs at various development stages in Caragana intermedia
  128. Cloning and expression analysis of SERK1 gene in Diospyros lotus
  129. Integrated metabolomic and transcriptomic profiling revealed coping mechanisms of the edible and medicinal homologous plant Plantago asiatica L. cadmium resistance
  130. A missense variant in NCF1 is associated with susceptibility to unexplained recurrent spontaneous abortion
  131. Assessment of drought tolerance indices in faba bean genotypes under different irrigation regimes
  132. The entire chloroplast genome sequence of Asparagus setaceus (Kunth) Jessop: Genome structure, gene composition, and phylogenetic analysis in Asparagaceae
  133. Food Science
  134. Dietary food additive monosodium glutamate with or without high-lipid diet induces spleen anomaly: A mechanistic approach on rat model
  135. Binge eating disorder during COVID-19
  136. Potential of honey against the onset of autoimmune diabetes and its associated nephropathy, pancreatitis, and retinopathy in type 1 diabetic animal model
  137. FTO gene expression in diet-induced obesity is downregulated by Solanum fruit supplementation
  138. Physical activity enhances fecal lactobacilli in rats chronically drinking sweetened cola beverage
  139. Supercritical CO2 extraction, chemical composition, and antioxidant effects of Coreopsis tinctoria Nutt. oleoresin
  140. Functional constituents of plant-based foods boost immunity against acute and chronic disorders
  141. Effect of selenium and methods of protein extraction on the proteomic profile of Saccharomyces yeast
  142. Microbial diversity of milk ghee in southern Gansu and its effect on the formation of ghee flavor compounds
  143. Ecology and Environmental Sciences
  144. Effects of heavy metals on bacterial community surrounding Bijiashan mining area located in northwest China
  145. Microorganism community composition analysis coupling with 15N tracer experiments reveals the nitrification rate and N2O emissions in low pH soils in Southern China
  146. Genetic diversity and population structure of Cinnamomum balansae Lecomte inferred by microsatellites
  147. Preliminary screening of microplastic contamination in different marine fish species of Taif market, Saudi Arabia
  148. Plant volatile organic compounds attractive to Lygus pratensis
  149. Effects of organic materials on soil bacterial community structure in long-term continuous cropping of tomato in greenhouse
  150. Effects of soil treated fungicide fluopimomide on tomato (Solanum lycopersicum L.) disease control and plant growth
  151. Prevalence of Yersinia pestis among rodents captured in a semi-arid tropical ecosystem of south-western Zimbabwe
  152. Effects of irrigation and nitrogen fertilization on mitigating salt-induced Na+ toxicity and sustaining sea rice growth
  153. Bioengineering and Biotechnology
  154. Poly-l-lysine-caused cell adhesion induces pyroptosis in THP-1 monocytes
  155. Development of alkaline phosphatase-scFv and its use for one-step enzyme-linked immunosorbent assay for His-tagged protein detection
  156. Development and validation of a predictive model for immune-related genes in patients with tongue squamous cell carcinoma
  157. Agriculture
  158. Effects of chemical-based fertilizer replacement with biochar-based fertilizer on albic soil nutrient content and maize yield
  159. Genome-wide identification and expression analysis of CPP-like gene family in Triticum aestivum L. under different hormone and stress conditions
  160. Agronomic and economic performance of mung bean (Vigna radiata L.) varieties in response to rates of blended NPS fertilizer in Kindo Koysha district, Southern Ethiopia
  161. Influence of furrow irrigation regime on the yield and water consumption indicators of winter wheat based on a multi-level fuzzy comprehensive evaluation
  162. Discovery of exercise-related genes and pathway analysis based on comparative genomes of Mongolian originated Abaga and Wushen horse
  163. Lessons from integrated seasonal forecast-crop modelling in Africa: A systematic review
  164. Evolution trend of soil fertility in tobacco-planting area of Chenzhou, Hunan Province, China
  165. Animal Sciences
  166. Morphological and molecular characterization of Tatera indica Hardwicke 1807 (Rodentia: Muridae) from Pothwar, Pakistan
  167. Research on meat quality of Qianhua Mutton Merino sheep and Small-tail Han sheep
  168. SI: A Scientific Memoir
  169. Suggestions on leading an academic research laboratory group
  170. My scientific genealogy and the Toronto ACDC Laboratory, 1988–2022
  171. Erratum
  172. Erratum to “Changes of immune cells in patients with hepatocellular carcinoma treated by radiofrequency ablation and hepatectomy, a pilot study”
  173. Erratum to “A two-microRNA signature predicts the progression of male thyroid cancer”
  174. Retraction
  175. Retraction of “Lidocaine has antitumor effect on hepatocellular carcinoma via the circ_DYNC1H1/miR-520a-3p/USP14 axis”
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