Genome-wide investigation and analysis of growth-regulating factor family in pineapple (Ananas comosus var. comosus)

Background

Growth-regulating factors (GRFs) are plant-specific transcription factors that play an important role in plant growth and development[1–5]. The most prominent feature of the GRF proteins is that it contains two highly conserved domains in N-terminal. The first is the QLQ domain (Gln, Leu, Gln), which is mainly rich in aromatic/hydrophobic amino acids. These conserved regions may play an important role in QLQ as a protein-protein interaction domain. For example, only fragments containing the QLQ domain can bind to the SNH domain in GIF[6]. The second is the WRC domain, which can bind to the C3H motif (Trp, Arg, Cys)[7].

Since the first GRF gene, OsGRF1, was cloned from Oryza sativa[8], A large number of GRF proteins have been identified from various plant species[3, 5, 7, 9–13]. GRF proteins play an important role in plant defense against various biotic and abiotic stresses[10]. A large number of GRF TFs were induced by abiotic stresses and take part in the regulation of plant tolerance to abiotic stress. For example, PhvGRFs may be involved in drought stress response and this response is species-specific[10]. UV-B radiation induces the accumulation of miRNA396, which is associated with reduced levels of GRF targets[14, 15]. AtGRF1 and AtGRF3 contribute to the regulation of various biological processes associated with defense response and disease resistance [16]. AtGRF7 acts as a repressor of a broad range of osmotic stress-responsive genes to prevent growth inhibition[17]. The expression of OsGRF1 in deep Oryza sativa internode meristems increased rapidly in response to GA and flooding[8].

GRF proteins are conserved during plant evolution, and the family is mainly expanded by gene duplication, especially large-scale duplication (WGD or segmental duplication)[18, 19]. The evolution of the GRF gene family provides new directions for understanding the evolution of biotic and abiotic stress responses and signaling in plants from simple unicellular to multicellular plants. Advances in genome sequencing technology have facilitated the evolutionary analysis of the GRF gene family, and genome-wide analysis of GRF genes has been performed in many species, which will contribute to further understanding of their evolutionary origin and biological function.

Pineapple is one of the world's three major tropical fruit trees and is the only commercially grown variety in the bromeliad family[20]. Abiotic stress is a major environmental condition that reduces plant growth, yield, and quality; in recent years, stress signaling research has made significant progress in many aspects[21]. Environmental stress significantly affected the growth and development of pineapple plants. High temperature caused sunburn in pineapple fruits, and low temperature caused slow growth of pineapple plants[20]. In addition, pineapple is also an important species for studying the evolution of monocots[22]. In this study, we identified GRF genes in pineapple and systematically analyzed their sequence characteristics, chromosomal distribution, phylogenetic relationships, gene structure, conserved motif composition, and collinearity. The expression of GRF gene family members in different biological processes and different tissue parts of pineapple was analyzed. This study provides valuable clues for the functional identification of the pineapple GRF gene family members.

Results

Identification and physicochemical properties of GRF protein in pineapple

Based on multiple sequence alignments and hidden Markov models of conserved domains QLQ and WRC, 21 candidate AcGRF genes were initially identified. Then, after using NCBI Conserved Domain Search and SMART to predict the QLQ and WRC conserved domains of AcGRFs, the sequences containing only one conserved domain were removed, and finally 8 genes were annotated as pineapple GRF genes. The validated AcGRF gene sequences were available in Additional file 1. After chromosomal location analysis, 8 GRF genes could be mapped on the linkage groups and were renamed from AcGRF1 to AcGRF8 based on their order on the linkage groups (Fig.1).

Gene characteristics, including coding sequence length, protein sequence length, exon number, protein molecular weight (MW), isoelectric point (PI), and subcellular localization results are listed in Additional file 2. Among the 8 AcGRF proteins, AcGRF4 containing 249 amino acids (aa) was the smallest protein, whereas the largest one was AcGRF6 containing 612 amino acids. All AcGRF proteins have molecular weights between 25.1KDa(AcGRF4)-99.3KDa(AcGRF7) and isoelectric points between 6.57(AcGRF5)-9.63(AcGRF4). The number of AcGRFs exons is between 2(AcGRF1)-5(AcGRF8). The predicted subcellular localization results showed that 7 AcGRF proteins were localized in the nucleus and one in the cell membrane (AcGRF4). Both the hydrophilicity and hydrophobicity results of the proteins were less than 0, indicating that these were all hydrophilic proteins.

Sequence and phylogenetic analysis of AcGRFs

The phylogenetic relationship of the AcGRF proteins was examined by multiple sequence alignment of their QLQ and WRC domains. In order to deeply study the evolutionary relationship among GRFs in pineapple, Oryza sativa, Arabidopsis, and Solanum Lycopersicum, a neighbor-joining phylogenetic tree was constructed using MEGA11 software. As shown in Figure 2a, QLQ and WRC domain sequences are highly conserved. All AcGRF proteins have the highly conserved sequence RTDGKKWRC, and 7 AcGRFs have a conserved QLQ (Gln-Leu-Gln) domain, the only exception being AcGRF5, whose QLQ domain composition is (Gln-Met-Gln).

The phylogenetic analysis(Fig.2b) indicated that the pineapple GRF proteins could be divided into three large groups(the group I, II, and III). Among the 8 AcGRFs proteins，3 belong to group I, 3 to group II, and 2 to group III. The GRF members in the group I can be further clustered into two subgroups (Ia and Ib), and group III can be further clustered into three subgroups(IIa, IIb, IIc). Among them, subgroup IIa is the largest, with 10 GRFs members, 6 monocots members, and 4 dicots members. Subgroup IIc is the smallest, containing only 4 dicot members; subgroup IIb contains 6 GRF members, 5 dicot members, accounting for 83%, and 1 monocot member, accounting for 17%, indicating that This subgroup of genes may have more important functions in dicots. In group III, there are 5 monocots members and 3 dicots members, with 62.5% of monocots members and 37.5% of dicots members, indicating that this family of genes may play a more important role in monocots. Monocots and dicots GRFs were almost evenly distributed in other subgroups, suggesting that these subgroups may have a similar status to monocots and dicots. The phylogenetic tree showed that AcGRFs were more closely related to OsGRFs, while AtGRFs were closely related to SlGRFs, which may be related to the fact that pineapple and Oryza sativa are monocots while Arabidopsis and Solanum Lycopersicum are dicots.

Unrooted phylogenetic trees of pineapple, Oryza sativa, Arabidopsis, and Solanum Lycopersicum GRF proteins. Phylogenetic tree constructed using the neighbor-joining method with a bootstrap value of 1000. Different colored arcs represent different families or subgroups. Different colors and fonts represent pineapple, Oryza sativa, Arabidopsis, and Solanum Lycopersicum GRF domains, respectively. GRF proteins from pineapple, Oryza sativa, Arabidopsis, and Solanum Lycopersicum are denoted by Ac, Os, At, and Sl prefixes, respectively.

Gene structure and motif composition analysis of AcGRFs

The exon-intron organizations of all the identified AcGRF genes were examined to gain more insight into the evolution of the GRF family in pineapple. As shown in Figure 3, all 8 AcGRF genes have intron-exon structures, and the number of exons varies between 2-5. AcGRF8 has the largest number of exons, AcGRF1 contains only two exons, and most AcGRFs genes contain 3-4 exons (Fig. 3d).

Protein motifs are the basic units of protein structure, which directly determine the function of a particular protein. To elucidate the diversity of AcGRF proteins, we used the online program MEME to analyze the conserved motifs of 8 AcGRF proteins (Fig. 3b and Additional file 3). There are between 4-10 conserved motifs of GRF proteins in pineapple. Motif1 and motif2 are present in all AcGRF proteins, and they represent the N-terminal WRC and QLQ-specific domains. For subgroup IIa members (AcGRF6 and AcGRF7), all Motifs were contained, while other group members contained varying amounts of Motifs. In conclusion, AcGRFs from within the same group has similar protein structures, although the number of motif arrangements is slightly different in some subgroup. Similar motif arrangements among AcGRF proteins suggest that protein structure is conserved within the specific subgroups. Gene structure and motif characteristics strongly support the phylogenetic relationship of AcGRFs.

synteny analysis of AcGRFs

Gene duplication plays an important role in promoting gene expansion, the three whole-genome duplications in Arabidopsis have been directly responsible for >90% of the increase in transcription factors, signal transducers, and developmental genes [23]. To gain a possible mechanism for the expansion of AcGRFs, we investigated gene duplication events in pineapple (Fig.4a). A total of two GRF pairs with duplication events (AcGRF8/Aco017250.1, AcGRF1/AcGRF2) were identified(Additional file 4), according to club’s definition of tandem duplication: a chromosomal region within 200kb containing two or more genes were defined as a tandem duplication event (Holub, 2001). Both duplication events identified were segmental duplications. This result suggests that fragment duplication plays a more critical role in the amplification of GRFs in the pineapple genome.

Given that comparative collinearity, maps are helpful for the study of evolutionary traits, we also established comparative collinearity maps of four species, Arabidopsis, Solanum Lycopersicum, Oryza sativa, and Musa, with pineapple (Fig. 4b). According to the results of collinearity analysis, we found 8 and 18 homologous gene pairs in monocots plants Oryza sativa and Musa, respectively, while 4 and 7 homologous genes were found in dicots plants Arabidopsis and Solanum Lycopersicum, indicating that the GRF gene has evolved over a longer period in pineapple, Arabidopsis and Solanum Lycopersicum, while it has undergone a shorter selective evolution with the GRF gene in Musa. Some AcGRFs were found to be collinear with multiple genes (especially between pineapple and Musa), such as AcGRF4 and AcGRF7, suggesting that these genes may play a major role in the expansion of the GRF gene family.

Interestingly, some genes, such as AcGRF4, were only colinear in the monocots plant Oryza sativa and Musa but not in the dicots plant Arabidopsis and Solanum Lycopersicum, suggesting that this gene was formed after the differentiation of monocots and dicots. Similarly, we also found that some genes (AcGRF2 and AcGRF7) were collinear in other species, suggesting that these genes may have existed before the differentiation of monocots and dicots plants. To understand the evolution of the GRF gene family more clearly, we successively calculated the Ka, Ks, and Ka/Ks of the gene pairs (Additional file 5). It may have experienced strong purifying selection pressure during evolution.

Identification and analysis of homeopathic elements in AcGRFs promoters

Cis-acting regulatory elements are important molecular switches in gene transcription regulation, involved in the regulation of gene transcription in plant growth and development and response to various biotic and abiotic stresses[24]. To further investigate the potential biological functions of AcGRFs genes in pineapple, we extracted the upstream 2000bp region sequences of all AcGRF genes and identified their homeopathic elements using the PlantCare online website. We analyzed the promoter sequences of eight AcGRFs genes. The results show that AcGRFs promoter elements are involved in auxin response, gibberellin response, abscisic acid response, jasmonic acid response, drought induction, meristem expression, endosperm expression, low-temperature response anaerobic induction, light response, etc. (Fig.5 Additional file 6), indicating that the AcGRF genes may be involved in a variety of stress and hormone responses. Among these elements, light-responsive elements accounted for the largest proportion (43.8% of all active elements), followed by anaerobic induction (14.2%), and abscisic acid and jasmonic acid responses also accounted for a larger proportion, 10.1%, and 9.5%, respectively. It suggests that AcGRFs may play an important role in light response and abscisic acid and jasmonic acid response, and may be involved in photomorphogenesis, plant senescence, and other processes, thereby regulating hypocotyl elongation, tissue and organ formation, stem elongation, and fruit ripening. Among the 8 AcGRFs, AcGRF8 has the most active elements (28), suggesting that it may be involved in more biological functions.

Expression patterns of AcGRFs in different tissues

To gain insight into the potential functions of GRF genes in pineapple, RNA-Seq data from 14 different pineapple tissue expression profiles corresponding to major stages of whole pineapple fruit development were used to create a heatmap of AcGRFs expression (Fig.6a, Additional File 7). We found that almost all AcGRFs had relatively high expression in various tissues early in development (eg, ovary, petals, receptacles, and sepals), revealing that AcGRFs may play a role in these tissues early in development. However, of the 8 pineapple GRFs, 5 GRFs were relatively highly expressed in the ovary and 1 was relatively highly expressed in the petals, suggesting that these genes play regulatory roles in the growth and development of these specific tissues.

To further analyze the expression patterns of GRF genes in different pineapple floral organs, we also created a heatmap of AcGRFs for 27 different pineapple floral organ samples (Fig. 6b, Additional file 7). The results showed that all genes except AcGRF3 had relatively high expression in the ovary and stamens, while AcGRF3 was relatively highly expressed in the pistil. Consistent with the previous transcriptome data, the more detailed transcriptome data also showed that AcGRFs had higher transcriptional levels in the early stages of development of various tissues and organs, further indicating that AcGRFs may play their functions in the early stages of growth and development.

Effects of Different Treatments on GRF Gene Expression in Pineapple

To further understand the effect of the pineapple GRF gene on biotic stress and hormone treatment, we used QRT-PCR to analyze the expression patterns of some pineapple GRF members under different treatments (Fig.7). Overall, some GRF genes were significantly induced or repressed by multiple abiotic factors. For example, under low-temperature treatment, the expression levels of AcGRF2 and AcGRF7 increased significantly at 8h and 12h and then gradually decreased, while the expression levels of AcGRF4 and AcGRF5 showed a decreasing trend after treatment, especially AcGRF5 was significantly increased at 2h after low-temperature treatment. reduce. During heat treatment, AcGRF2, AcGRF4, AcGRF5, and AcGRF7 were significantly reduced in different periods, especially AcGRF2 and AcGRF7, and the difference in expression reached a significant level 2h after treatment. For gibberellin treatment, the expression levels of AcGRF2, AcGRF4, and AcGRF7 were significantly different after 2h, while AcGRF5 was only significantly different at 8h. These results indicated that AcGRFs were involved in phytohormone and abiotic stress responses to varying degrees.

Discussion

The GRF gene family is a small family of transcription factors with important functions in plant specificity[18]. GRF family members play important roles in biotic and abiotic stress situations. Several studies have revealed the function of GRF family genes[25–32]. In this study, we performed the first genome-wide identification and analysis of GRFs in pineapple. Eight GRF members were identified in the pineapple genome and named AcGRF1 to AcGRF8 according to their chromosomal locations.

Based on the analysis of the conserved domains of pineapple GRF proteins, it was found that the QLQ and WRC domains of most GRF proteins were conserved. A similar situation was found in Arabidopsis, where the leu in AtGRF9 was replaced by Phe[27]. Based on phylogenetic tree analysis, we divided the 8 GRF genes into 6 subgroups. It was found that Oryza sativa and pineapple GRF genes are more closely related, while Arabidopsis and Solanum Lycopersicum are more closely related, which may be related to the differentiation of monocots and dicots plants.

Gene duplications are one of the primary driving forces in the evolution of genomes and genetic systems[33]. There is evidence for at least two ancient whole-genome duplication events in pineapples. The first was a τ event, which occurred about 135 to 110 million years ago, and the second whole-genome doubling event, a σ event, occurred about 120 to 100 million years ago[22]. By comparing the collinearity of GRF genes in Oryza sativa, Musa, Solanum Lycopersicum, Arabidopsis, and pineapple, we found that Oryza sativa, Musa, and pineapple have strong collinearity, but there is less collinearity with Solanum Lycopersicum Arabidopsis, which further proves that Oryza sativa, Musa and pineapple are closely related, while Solanum Lycopersicum and Arabidopsis are closely related. It also indicates that the number of GRF gene family members in pineapple is less than that of Oryza sativa (13) and Musa (19), which may be because pineapple has fewer whole-genome duplication events than Oryza sativa (3 times) and Musa (4 times) and gene loss in whole gene duplication.

Studies have shown that the expression level of the GRF gene in young tissues is significantly higher than that in mature tissues[11, 19, 27, 34]. For example, GmGRFs have strongly expressed in shoot apical meristems, developing seeds and flowers[34]. The expression profiles of AcGRF genes were analyzed from the transcriptome data of two different varieties of pineapples, and it was shown that most of the AcGRF genes were relatively highly expressed in the early developmental stages of various tissues and organs, but were weakly expressed in mature tissues. The promoter analysis of AcGRFs found that AcGRF2, AcGRF5, and AcGRF6 have meristem expression elements, indicating that these genes may play an important role in the growth tissue of pineapple.

Plants cannot move, so they must endure abiotic stresses such as drought, salinity, and extreme temperatures, To withstand environmental stresses, plants have evolved interconnected regulatory pathways that enable them to respond and adapt to their environments promptly [35].In previous studies, the introduction of many stress-inducible genes via gene transfer resulted in improved plant stress tolerance[36, 37]. For example, AtGRF7 mutants are more tolerant to salt and drought stress than wild-type and overexpression lines[38]. AtGRF1 and AtGRF3 may play a central coordinating role in plant growth and defense[18]. In our study, 4 of the 8 AcGRF promoter homeopathic elements contained stress-responsive elements and drought-responsive elements, respectively. RNA-seq data showed that AcGRFs had relatively high expression in the early developmental stages of various tissues, revealing that AcGRFs may play a role in the early developmental stages of these issues. qRT-PCR results showed that AcGRFs were involved in phytohormone and abiotic stress responses to varying degrees. But more experimental evidence is needed on how they respond to these abiotic stresses in pineapple.

Methods

Identification of Pineapple GRF Gene

To identify the pineapple GRF gene, the pineapple genome sequence and annotation files were downloaded from the phytozome database (https://phytozome-next.jgi.doe.gov/) and downloaded separately from the Uniport database (https://www.uniprot.org). Nine identified GRF protein sequences in Arabidopsis thaliana and 12 GRF protein sequences identified in Oryza sativa were used to call their GRF protein sequences as queries using TBtools to perform a local BLASTP search on the pineapple genome[39]. Furthermore, to identify AcGRF proteins that may have been missed during the search by BLASTP, Hidden Markov Model (HMM) files corresponding to the conserved domains of GRF QLQ (PF08880) and WRC (PF08879) were downloaded from the Pfam protein database(https://pfam.xfam.org/). The TBtools plug-in Simple HMM Search was used to locally search for sequences containing WRC and QLQ domains from the pineapple genome sequence based on HMM, and the E-value was set to 1e-10. Next, using Batch CD-Search and SMART programs to confirm the presence of the GRF core sequence, all candidate genes that may contain QLQ and WRC domains in the search results by BLASTP and HMMER were further examined. Each candidate gene was then manually checked to ensure a conserved heptapeptide sequence at the N-terminal between the predicted QLQ and WRC domains. The ExPASy-ProtParam database (https://web.expasy.org/protparam/) was used to analyze the physicochemical properties of the AcGRF genes, such as coding region length, number of amino acids, molecular weight (MW), theoretical isoelectric point (PI). Use the website (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/#), (http://www.cbs.dtu.dk/services/TMHMM/), (http:// www.cbs.dtu.dk/services/SignalP/) to predict the subcellular location, transmembrane domain and signal peptide prediction of AcGRFs, respectively.

AcGRFs gene sequence alignment and phylogenetic analysis

Multiple alignments were performed using MEGA using all identified pineapple GRF protein sequences with Oryza sativa, Arabidopsis, Musa, and Solanum Lycopersicum GRF protein sequences. All identified AcGRF genes were divided into different families according to the OsGRF, AtGRF classification scheme, and the alignment of AcGRF with the conserved domains of OsGRF, and SlGRF proteins. The phylogenetic tree adopts the neighbor-joining method of Mega11, using parameters: Poisson model, pairwise deletion, and 1000 bootstrap replicates. Use itol (https://itol.embl.de/) for visualization and beautification.

Analysis of AcGRFs structure, conserved motifs, gene promoters, and functional elements

The exon-intron structures of all AcGRFs genes were analyzed using the TBtools plugin Visualize Gene Structure. Check for conserved motifs by submitting all full-length AcGRF gene protein sequence files to the MEME online program (https://meme-suite.org/) with the following parameters: site distribution is set to zero or one occurrence per sequence, motif number is set to 10. The 2Kb sequence upstream of the translation initiation codon of the AcGRF gene was extracted using TBtools, and the sequence was submitted to Plantcare (https://bioinformatics.psb.ugent.be/) to predict the cis-acting elements of the AcGRF gene.

Chromosome distribution and evolution analysis of AcGRFs

Using TBtools software, all AcGRF genes were mapped to pineapple chromosomes according to the physical location information in the pineapple genome database[39]. Gene duplication events were analyzed using the multicollinearity scan tool (MCscanX) using default system parameters. To reveal the collinearity of homologous GRF genes obtained in pineapple and other species, a collinearity map was constructed using the TBtools plugin dual Systeny Polt. Non-synonymous (Ka) and synonymous (Ks) substitutions of AcGRF for each repeat were calculated using the KaKs Calculator.

Plant materials and treatments

Ananas comosus cv. Shenwan, a typical cultivated variety, was used throughout the study. RNA-seq data from NCBI under Project ID PRJNA483249 and PRJEB38680, respectively. To investigate the expression pattern in response to various stress and hormonal treatments, several AcGRF genes were selected for further qRT-PCR analysis. For cold and heat treatments, pineapple seedlings were subjected to immersion at 4°C and 42°C for 0, 2, 4, 8, 12, 24, 48, and 72 h, respectively. The leaves were collected at 2, 4, 6, 8, 10, 12, and 24h in cold treatment and 4, 8, 12, 24, and 48 h in heat treatment. All treated tissue samples were immediately frozen in liquid nitrogen and stored at − 80 °C for subsequent analysis.

RNA extraction and gene expression analysis

Total RNA was extracted using the procedure of CWBIO RNApure Plant Kit (DNase I). RNA quality was checked using agarose gel electrophoresis and RNA concentration was subsequently calculated using Nanodrop ND-1000. According to the manufacturer's protocol, First-strand cDNA was synthesized using DNA-free RNA using the HiScript® II 1st Strand cDNA Synthesis Kit (Vazyme). Quantitative RT-PCR was performed using a Roche Lightcyler® 480 instrument using SYBR Green chemistry. The housekeeping pineapple β-actin gene was used as an internal control. Reaction conditions: 95°C for the 30s, followed by 40 cycles of 95 °C, /10 s, 60 °C, /30 s. Each reaction was performed in biological triplicates and the data from real-time PCR amplification was analyzed using the 2⁻^△△^CT method. The primer sequences used in this study are shown in detail in the Additional file8. Transcriptome data from Mao et al[40]; Wang et al[41]. Transcript abundance of pineapple GRF genes was calculated as Transcripts Per Kilobase of exon model per Million mapped reads. Transcriptome data used in this study were obtained from NCBI projects PRJNA483249 and PRJEB38680.

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Genome-wide investigation and analysis of growth-regulating factor family in pineapple (Ananas comosus var. comosus)

Abstract

Background

Results

Discussion

Conclusions

Methods

Abbreviations

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References