Skip to main content
Download PDF

Identification of ARF family in blueberry and its potential involvement of fruit development and pH stress response

BMC Genomics volume 23, Article number: 329 (2022) Cite this article

Abstract

Background

Auxin responsive factor (ARF) family is one of core components in auxin signalling pathway, which governs diverse developmental processes and stress responses. Blueberry is an economically important berry-bearing crop and prefers to acidic soil. However, the understandings of ARF family has not yet been reported in blueberry.

Results

In the present study, 60 ARF genes (VcARF) were identified in blueberry, and they showed diverse gene structures and motif compositions among the groups and similar within each group in the phylogenetic tree. Noticeably, 9 digenic, 5 trigenic and 6 tetragenic VcARF pairs exhibited more than 95% identity to each other. Computational analysis indicated that 23 VcARFs harbored the miRNA responsive element (MRE) of miR160 or miR167 like other plant ARF genes. Interestingly, the MRE of miR156d/h-3p was observed in the 5’UTR of 3 VcARFs, suggesting a potentially novel post-transcriptional control. Furthermore, the transcript accumulations of VcARFs were investigated during fruit development, and three categories of transcript profiles were observed, implying different functional roles. Meanwhile, the expressions of VcARFs to different pH conditions (pH4.5 and pH6.5) were surveyed in pH-sensitive and tolerant blueberry species, and a number of VcARFs showed different transcript accumulations. More importantly, distinct transcriptional response to pH stress (pH6.5) were observed for several VcARFs (such as VcARF6s and VcARF19-3/19–4) between pH-sensitive and tolerant species, suggesting their potential roles in adaption to pH stress.

Conclusions

Sixty VcARF genes were identified and characterized, and their transcript profiles were surveyed during fruit development and in response to pH stress. These findings will contribute to future research for eliciting the functional roles of VcARFs and regulatory mechanisms, especially fruit development and adaption to pH stress.

Peer Review reports

Background

Blueberry (Vaccinium spp.) is an economically important small fruit crop widely cultivated all over the world. Blueberry fruit tastes sweet with variable acidity. More importantly, its fruit possesses human health-promoting effects due to the richness of active compounds (such as flavonoids, phenolic acids, and anthocyanins, which have been known as potent antioxidants), for example, vision improvement, anti-cancer activity, aging delay, and reduced risk of cardiovascular diseases [1]. Thus, considerable attention has been attracted to understand the regulation of its growth and development. To date, a number of players have been addressed to perform important functions in blueberry, including transcription factors (such as VcSPL12, VcRR2, VcMYBs, VcFT), miRNAs, and auxin-related gene VcIAA27 [2,3,4,5,6,7]. Recently, high-throughput sequencing data provided important information for understanding the regulators that control blueberry growth and development [8,9,10]. However, our understandings of the regulatory network underlying its growth and development are extremely limited. More regulators need to be comprehensively identified and functionally addressed in blueberry.

The phytohormone auxin is involved in the regulation of almost all developmental processes in plants, and it governs gene expression through auxin signalling pathway. Auxin responsive factor (ARF) family is considered as one of core components in auxin signalling pathway [11]. Generally, ARF proteins comprise a DNA binding domain (DBD) at its N-terminal, an activation or repression domain at its middle region (MR), and dimerization domain (CTD) at its C-terminal [12]: the DBD is a critical domain for the recognition of the auxin-responsive element (AuxRE); the MR domain determines transcriptional activation or repression; the CTD domain is involved in the regulation of ARF activity by dimerizing with Aux/IAA family genes as well as between ARFs. Under low auxin levels, ARFs are generally under inactive status through formatting a complex with their inhibitor Aux/IAA proteins, therefore blocking the auxin signalling pathway. When auxin levels increase, however, Aux/IAA proteins can be directly bound by the SCF (TIR1/AFB) ubiquitin ligase, followed by protein degradation at the 26S proteasome. After release from Aux/IAA inhibition, ARFs can then activate or repress the expression of auxin-dependent genes. Thus, the Aux/IAA-ARF modules govern diverse processes of plant growth and development such as apical dominance, later root initiation and formation, and vascular differentiation, hypocotyl xylem expansion and cambium homeostasis, fruit development and maturation, cell division, expansion, and differentiation [11, 12]. For example, AtARF4 in Arabidopsis acts as a transcriptional repressor to regulate shoot regeneration through competing the interaction of AtIAA12 with AtARF5 [13]; MdARF13 in apple serves as a negative regulator of the anthocyanin metabolic pathway [14]; AtARF7 and AtARF19 confer the gravitropism and phototropism of plant hypocotyls through mediating the asymmetric expression of AtSAUR genes in Arabidopsis [15]; MdARF8 facilitates lateral root formation in apple [16]. Noticeably, the Aux/IAA-ARF modules perform their functions in not only organ-dependent but also cell type-dependent way. For instance, the two complexes, AtJMJ30-AtARF and AtATXR2-AtARF, can alter the status of H3K9me3 and H3K36me3 at the loci of AtLBDs, respectively, therefore promoting their expressions during leaf-to-callus transition in Arabidopsis [17, 18]; tasiR-ARFs inhibits the expression of AtARF3 in the hypodermal cells of Arabidopsis to repress the fate of ectopic megaspore mother cell [19]; SlARF3 is involved in the formation of epidermal cells and trichome in tomato [20]; AtARF5/MP and its inhibitor AtIAA12/BDL enable to specify root cell in Arabidopsis [21].

As a critical component of the auxin signaling pathway, ARFs are encoded by a multigene family. The first ARF gene was isolated in Arabidopsis using a yeast one-hybrid screen, and several other ARFs were subsequently identified by sequence homology search or Y2H screen [22]. With the availability of genome and transcriptome sequences, ARF family have been identified and characterized in a number of plant species. For example, 31 ARF genes were identified in apple [23], 19 in grape and sweet orange [24, 25], 39 in poplar [26], 17 in physic nut [27], 89 in the three Apiaceae species (celery, coriander, and carrot) [28], 21 in tomato [29], and 25 in rice [30]. Emerging genetic and molecular evidences indicated that ARF family is likely responsible for the specificity of the auxin responses, and the distinct properties among ARF family might facilitate the generation of unique auxin response that triggers developmental process appropriately [31, 32]. The most direct evidence is that a number of ARF genes can serve as transcriptional activators, and some as repressors instead [33]. For instance, three MpARFs were identified in Marchantia polymorpha, and MpARF1 and MpARF2 were transcriptional activator and repressor, respectively, whereas MpARF3 failed to show the activity of transcriptional activator and repressor [34]. Recently, DAP-Seq data provided a framework for understanding both specific and redundant aspects of ARF binding [35]. Thus, the identification and characterization of ARF family in distinct plant species is an essential step towards understanding their functional roles.

In blueberry, it has been reported that auxin might function in several processes such as the development of adventitious root, fruit growth, shoot proliferation, and leaf formation [4, 10, 36]. However, a comprehensive analysis of blueberry ARF (VcARF) family has not yet been reported. In the present study, 60 VcARF genes were genome-widely identified and characterized. Their gene structures, motif architectures, chromosomal locations, phylogenetic relationship were investigated. Furthermore, the miRNA responsive elements and cis-acting elements was computationally analyzed, and the transcript profiles of VcARFs were retrieved from publicly available data during fruit development and in response to pH stress. The findings will contribute to future research for addressing the functional roles of VcARF family, especially fruit development and adaption to pH stress.

Results

Characterization of ARF family genes in blueberry

To identify ARF family in blueberry, the CDS sequences of the ARF genes from apple, grape, tomato, Arabidopsis and rice were used as queries to conduct BLASTn against the database Vaccinium corymbosum GDV RefTrans V1. After the removal of redundant sequences, 60 VcARF genes were totally identified, which were correspondingly named in term of their homologs in Arabidopsis (Fig S1). It was reported that Arabidopsis ARFs were classified into five groups [37]. Noticeably, no VcARF was clustered into the class IV with inclusion of AtARF12-15 and AtARF20-23 (Fig S1).

The detailed information of all the 60 VcARFs is listed in Table S1. Briefly, the CDS lengths of VcARF family vary from 1698 to 6417, and their deduced proteins comprise 565–2138 amino acids with the estimated molecular weights from 62.77 to 236.27 kDa and the predicted protein isoelectric points from 5.46 to 8.61. All the VcARF proteins contain an Auxin_resp domain and a N-terminal B3-type DNA binding domain (DBD) that recognizes the auxin-responsive element (AuxRE), supporting that they are ARF proteins. The prediction of subcellular localization indicated that most of VcARF proteins are completely or mainly localized in the nuclear, whereas three large VcARFs are predominately distributed in the cytoplasm (VcARF2-2/2–3) or plasma membrane (VcARF2-1).

Chromosomal localization of VcARF family genes and their evolutionary relationships

To investigate the distribution of VcARF family genes in the draft genome (1760 scaffolds), a Circos map was built using the corresponding scaffolds where the VcARF genes are localized. Consequently, VcARFs are unevenly situated in 40 distinct scaffolds. As shown in Fig. 1, twenty five scaffolds harbor one ARF gene, while two ARF genes are separately distributed in each of 10 scaffolds and three ARFs in each of 5 scaffolds. Gene family can be generated from tandem duplication and segmental duplication of chromosomal regions as well as whole-genome duplication (WGD) [38, 39]. Tandem duplication is generally defined as two paralogs separated by the distance of 200 kb or less in the same chromosome [39]. It was observed that two pairs of VcARFs (VcARF19-1 and VcARF19-3; VcARF9-2 and VcARF9-5) are separated by 46.7 kb and 55.5 kb, respectively (Fig. 1). The identities at nucleotide level are separately 88% and 80% between VcARF19-1 and VcARF19-3 as well as VcARF9-2 and VcARF9-5 (Table S2), suggesting that the gene pairs might be derived from tandem duplication. Meanwhile, it was noticed that 9 digenic, 5 trigenic and 6 tetragenic VcARF pairs show very high identity (more than 95%) to each other (Fig. 1 and Table S2), implying that they were possibly generated from segmental or whole-genome duplication. Furthermore, the non-synonymous/synonymous substitution ratio (Ka/Ks) was calculated between duplicated gene pairs. As shown in Table S2, the Ka/Ks values of all the gene pairs are less than 1 with the exception of two gene pairs (VcARF19-1 and VcARF19-3, VcARF18-2 and VcARF18-5), suggesting that these gene pairs and trigenic VcARFs have possibly undergone a purifying selection with restricted functional diversification.

Fig. 1
figure 1

Chromosomal localization of the VcARF family genes. Each colored box represents a scaffold. The approximate distribution of each VcARF gene is marked on the circle with a short black line. The tandem duplicated genes are indicated with stars. Colored lines refer to the linkage group with high identity, red line 100%; blue line 99–100%; green line 98–99%; yellow-green 95–98%

Full size image

To investigate their evolutionary relationship, phylogenetic analysis was performed using the CDS sequences of VcARFs and the ARFs from grape, apple, tomato, Arabidopsis and rice. Consequently, all the ARFs were classified into 5 different groups. VcARFs were separately distributed to the 5 groups and clustered together with ARFs from other plant species (Fig. 2). This observation suggested that VcARF family might have undergone evolutionary diversification similar to their counterparts from the other four plant species. Further observation indicated that a number of VcARFs were distributed at the same clade with function-known ARFs. For example, VcARF5s was grouped with SlARF5 in the phylogenetic tree (Fig. 2), which affects fruit set and development through regulating auxin and gibberellin signaling in tomato [40]; loss of function of SlARF4 clustered with VcARF4s confers the tolerance of water deficit in tomato [41].

Fig. 2
figure 2

Phylogenetic relationship of VcARF genes with the ARFs in other plant species. The sequences of publicly known ARF genes in Arabidopsis (23), apple (28), grape (26), tomato (25) and rice (25) were downloaded from NCBI (www.ncbi.nlm.nih.gov), TAIR (www.arabidopsis.org) or RAP-DB (https://rapdb.dna.affrc.go.jp/) database. A maximum likelihood tree was generated with the CDS sequences of 187 ARF genes using the MEGA7 software, which were clustered into 5 groups (I-V). The numbers indicate the bootstrap values, which were calculated using 1000 replicates. The ARFs in the same species are represented with the same symbol: blue diamond, Vaccinium corymbosum; purple triangle, Vitis vinifera; dark purple triangle, Malus domestica; red triangle, Solanum lycopersicum; green triangle, Arabidopsis thaliana; light green triangle, Oryza sativa

Full size image

VcARF family shows diverse gene structures and motif compositions

Since the structural diversity provides important clues for gene duplication within gene family, the exon/intron structures were generated in terms of the gene coding and genomic sequences. It was observed that VcARF genes show a high variation in the number of exons. As shown in Fig. 3 and Table S1, forty two VcARFs have 10–16 exons with intron intervals. Fourteen VcARFs comprise only 2–4 exons including all the VcARF16s and VcARF17s, whereas a large number of exons exist in the genes VcARF2-1, VcARF2-2, VcARF2-3 and VcARF9-2 with the number of 32, 23, 25, 20, respectively (Fig. 3). Furthermore, integration analysis of phylogenetic relationship with gene structures was performed. Consequently, the VcARF pairs at the same clade in phylogenetic tree basically show similar exon/intron structures with high identity of more than 85% (Fig. 3). These observations supported that the VcARF pairs at the same clade might contribute to gene family expansion with less functional diversification.

Fig. 3
figure 3

The exon/intron structures of VcARF genes. The left panel is the phylogenetic tree of VcARF genes. Three groups are clustered (I-III), and the identity between the gene pair is listed. The right panel shows the intron–exon structures where the exons are shown by blue rectangle, and the introns are represented by thin lines. Light green rectangle refers to upstream/downstream (5’UTR and 3’UTR)

Full size image

To provide information regarding the functional diversity of VcARF family, the components of their motifs and domains were analyzed using the online tools MEME and Pfam. As a result, all the family members contain B3-type and Auxin_resp domains (Fig. 4). However, 27 VcARF proteins mainly at the class II and III lack the C-terminal AUX/IAA or PB1 domain functioning as a dimerization domain with AUX/IAA proteins, including VcARF3s, VcARF4s, VcARF16s, VcARF17s, VcARF2-1/2–2/2–3/2–4, VcARF8-1/8–2, VcARF19-1/19–2 (Fig. 4). This observation is consistent with previous reports that C-terminal AUX/IAA is missed in several ARF proteins in plants [28, 29, 37, 42]. Notably, additional domains were observed in five VcARF proteins (VcARF2-1/2–2/2–3/2–4/9–2). For example, VcARF2-1/2–2/2–3 contain Clp_N, ClpB_D2-small and three AAA-related domains which are related to binding, oligomerization or chaperone-like functions, while VcARF9-2 harbors Ketoacyl-synt_C and Ketoacyl-synt domains where the active site of beta-ketoacyl synthase is usually located (Fig. 4). Meanwhile, 20 motifs were identified for the 60 VcARFs. Although most of the VcARF proteins share the motifs 1, 2, 3, 4, 5, 6, 8, 10, diverse motif compositions exist in VcARF family (Fig. 4), implying their functional diversity. Further observation indicated that VcARF proteins at the same clade in the phylogenetic tree basically show similar motif composition (Fig. 4), suggesting a potential of functional redundancy.

Fig. 4
figure 4

The conserved domains and motif compositions of VcARF proteins. (A) The phylogenetic tree of VcARF proteins, and three groups are clustered (I-III). (B) The conserved domains of VcARF proteins. Different colored rectangles represent different domains. (C) The motif compositions of VcARF proteins. Twenty motifs were identified using the program MEME, which are represented by the boxes of different colors. (D) The sequence logos of 20 motifs. The height of the letters within each stack indicates the relative frequency. The symbols are corresponding to the colored box in the right panel

Full size image

A group of ARF genes are targeted by miRNAs in blueberry

MicroRNAs plays important roles in the regulation of gene expression through mRNA cleavage or translational repression in plants. It has been well acknowledged that ARF genes can be targeted by miR160 and miR167 in plants [32]. To explore the VcARF family members potentially targeted by miRNAs, the miRNA responsive elements (MREs) were searched using the ORF sequences of 60 VcARFs against all the vco-miRNAs which were identified previously [3]. Consequently, 14 VcARFs (12 VcARF16s and 2 VcARF17s) harbor one MRE for miR160, while all the 6 VcARF8s and 3 VcARF6s (VcARF6-1/6–2/6–4) show one MRE for miR167 at the coding region (Fig. 5), which is in agreement with the previous reports that ARF6/8/16/17 can be targeted by miR160 or miR167 [43, 44]. These observations suggested that they might have the potentials to be targeted by miR160 or miR167. Interestingly, the MRE for miR156d/h-3p is present in the 5’UTR of 3 VcARFs such as VcARF9-4, VcARF9-5, VcARF19-4 (Fig. 5), suggesting that the post-transcriptional regulation of ARFs mediated by miR156-3p possibly exists in blueberry.

Fig. 5
figure 5

The diagrams of VcARF sequences targeted by miR167, miR160 or miR156. The orange box, black line, blue box, and green box represent CDS sequence, UTR region, ARF domain, and miRNA responsive element (MRE), respectively. The numbers indicate the localization of each MRE in the CDS sequence. Mature miRNA sequences are aligned along with their complementary sequences within VcARF genes, and the matched nucleotides are labeled with double dots

Full size image

VcARF family shows diverse transcript profiles during fruit development

To obtain hints for understanding the regulatory mode of gene expression, the cis-acting elements were predicted in the promoter regions of VcARFs (1500 bp upstream of the TSS) using the online program PlantCARE. As indicated in Fig. 6, besides the core elements, the VcARF family show 48 types of specific-function elements, including development-associated elements (for example RY-element, GCN4_motif, HD-ZIP), light responsive elements (such as G-box, TCT-motif, GT1-motif, AE-box, Box 4, GATA-motif, I-box, TCCC-motif, MRE, 3-AF1 binding site, chs-CMA1a, Box II, ATCT-motif, ACE, chs-CMA2a, Gap-box, Sp1), stress response elements (for example ARE, MBS, TC-rich repeats, LTR, GC-motif), phytohormone response element (such as ABRE, GARE-motif, P-box, TATC-box, TGACG-motif). Furthermore, phylogenetic analysis was performed using their promoter sequences. Interestingly, a number of promoters belonging to homologous VcARFs were clustered at the same clade in the phylogenetic tree, showing similar compositions of cis-acting elements (for example VcARF16-5/16–6/16–7, VcARF9-3/9–4/9–5) (Fig. 6). Generally, TGA-element and AuxRR-core are considered as auxin-related elements. It was observed that 25 VcARFs have the element TGA-element in their promoter regions, while the element AuxRR-core is present in the promoter regions of VcARF9-1, VcARF9-3 and VcARF9-4 (Fig. 6), supporting that they might be responsive to auxin activation or repression.

Fig. 6
figure 6

The cis-acting elements in the promoter regions of VcARF genes. The phylogenetic tree was generated using the promoter sequences of VcARF genes (the left panel), and the colored boxes in right panel represent different cis-acting elements. The symbols at the bottom are corresponding to the colored box in the right panel

Full size image

Blueberry fruit development can be generally divided into two phases: fruit growth and maturation. To understand the expression patterns of VcARFs during fruit development, we extracted the transcript profiling data of fruit at differently developmental stages including pad, cup, mature green, pink and ripe from the previous study reported by Gupta et al. [45]. The three early developmental stages represent the growth phase; while the pink and blue stages refer to the maturation phase. Consequently, the transcript profiling data of 44 VcARFs were obtained. As shown in Fig. 7A, they can be grouped into 3 classes based on their accumulation patterns. The class I comprises 9 VcARFs with relatively high transcript abundance at maturation stage. Notably, VcARF2-2 and VcARF2-3 were relatively low at the three early developmental stages, but then remarkably increased from pink to ripe stages (Fig. 7A). The class II consists of 29 VcARFs including VcARF1s, VcARF4s, VcARF7s, VcARF8s, VcARF9s, VcARF17-2, VcARF2-5/2–6/2–7/2–8, VcARF16-5/16–6/16–7/16–8, which were highly expressed at the pad stage and gradually decreased as fruit develops (Fig. 7A). The class III contains 6 VcARFs with relatively high accumulation at either cup or mature green stage such as VcARF18s and VcARF2-1/2–4 (Fig. 7A). These transcript profiles implied that VcARF family might play different, even opposite, roles during fruit development.

Fig. 7
figure 7

Expression analysis of VcARF genes during fruit development. A Transcript profiling of VcARFs during fruit development. The transcriptome data from different developmental stages (pad, cup, mg, pink, ripe) were extracted from the previous study reported by Gupta et al. [45]. The color scale beside the heat map indicates gene expression levels, low transcript abundance indicated by blue color and high transcript abundance indicated by red color. B Photograph of fruits at five developmental stages [green pad (GPS), green cup II (GCS), light green/white (FWS), pink (FPS) and blue (FMS) fruits], the scale bar indicates 1 cm. C-E Expression patterns of VcARFs during fruit development. Total RNAs were extracted from fruit of the cultivar ‘Northland’ (V. corymbosum) at the five above-mentioned developmental stages. Data were normalized against VcACTIN, and the expression level at GPS stage was set as 1. Error bars indicate SE of three biological and technical replicates, and different letters indicate significant difference (P < 0.05)

Full size image

Subsequently, ten VcARFs were chosen to investigate their expression patterns at five fruit developmental stages [green pad (GPS), green cup II (GCS), light green/white (FWS), pink (FPS) and blue (FMS) fruits] (Fig. 7B) using qRT-PCR approach, including VcARF1-1, VcARF2-1, VcARF2-5, VcARF4-1, VcARF6-1, VcARF7-1, VcARF8-4, VcARF16-1, VcARF18-1. As shown in Fig. 7C, the expressions of VcARF2-1, VcARF4-1, VcARF8-4 and VcARF9-1 were decreased as fruit develops, whereas VcARF16-1 was transcriptionally increased when fruit ripens (Fig. 7D). Additionally, two genes VcARF1-1, VcARF2-5 showed relatively high expression at GPS and FPS stages (Fig. 7D), while maximum expression was observed at the FWS stage for the three genes VcARF1-1, VcARF2-5, VcARF6-1 (Fig. 7E).

ARF family exhibits different expression patterns in response to pH shift in blueberry

Blueberry prefers to grow in acidic soils within the pH range from 4.0 to 5.3, and neutral to basic soil pH is usually harmful for its growth [36]. To explore the roles of VcARF family in response to pH stress, we retrieved the publicly available transcript profiling data of blueberry root (two species: pH-sensitive Vaccinium corymbosum and pH-tolerant Vaccinium arboretum) at its preferred pH of 4.5 and near neutral pH 6.5. According to their transcript accumulation patterns, the VcARFs in both species were classified into four groups (Fig. 8). The VcARFs in the groups I and II showed relatively low and stable expression in different pH conditions. However, altered transcript accumulation was observed for the VcARFs in the group III and IV (Fig. 8). In pH-sensitive V. corymbosum, VcARF1s and VcARF18s showed relatively high transcript accumulation under the condition of preferred pH (4.5), whereas relatively high accumulation was observed for VcARF3s, VcARF19-3 and VcARF19-4 at near neutral pH 6.5 (Fig. 8A). In pH-tolerant V. arboretum, the VcARFs in the group III and IV (such as VcARF1s, VcARF6s, VcARF6s, VcARF18s, VcARF2-5/2–6/2–7/2–8, VcARF19-3/19–4) were suppressed by near neutral pH 6.5 (Fig. 8B). When compared the accumulation patterns of VcARF genes between pH-sensitive and pH-tolerant species, it was found that VcARF1s and VcARF18s displayed similar accumulation patterns in both species (Fig. 8). Interestingly, several VcARFs showed different, even opposite, accumulation patterns. For example, VcARF6s and VcARF19-3/19–4 in pH-tolerant V. arboretum were depressed when the pH shifted from 4.5 to 6.5 (Fig. 8B), whereas the converse patterns were observed for them in pH-sensitive V. corymbosum (Fig. 8A).

Fig. 8
figure 8

Expression analysis of VcARF genes in response to pH stress. A-B Transcript profiling of VcARFs in response to pH stress. The transcriptional response to different pH conditions (pH4.5 and pH6.5) in pH-sensitive Vaccinium corymbosum (A) and pH-tolerant Vaccinium arboretum (B). The transcriptome data were extracted from the publicly-available GDV database (http://www.vaccinium.org) for heatmap generation. Each of pH treatment has four samples for each species, and the numbers represent the sample IDs. The color scale above the heat map indicates gene expression levels, low transcript abundance indicated by blue color and high transcript abundance indicated by red color. C-D Expression patterns of VcARFs in response to pH stress. Total RNAs were extracted from tissue culture seedlings of the cultivar ‘Northland’ (V. corymbosum) under pH 4.8, 5.2, 5.8, 6.8. Data were normalized against VcACTIN, and the expression level under pH 4.8 was set as 1. Error bars indicate SE of three biological and technical replicates, and different letters indicate significant difference (P < 0.05)

Full size image

Increasing evidence indicated that above-ground plant parts can quickly respond to pH stress via regulating its growth and signal exchange between leaf and root [46]. To explore how ARF genes affect the adaption of above-ground tissues to pH switch, the young leaves of tissue culture seedlings were used to investigate their responses to pH shift (pH 4.8, 5.2, 5.8, 6.8) in V. corymbosum. Ten VcARFs were chosen to perform qRT-PCR analysis, including VcARF6-1, VcARF7-1, VcARF16-1, VcARF9-1 and six genes with publicly available transcript profiling (VcARF1-1, VcARF2-1, VcARF2-5, VcARF4-1, VcARF8-4, VcARF18-1). As shown in Fig. 8C, seven genes VcARF1-1, VcARF2-1, VcARF2-5, VcARF6-1, VcARF8-4, VcARF16-1, VcARF18-1 were transcriptionally decreased as pH shifted from 4.8 to 6.8. Especially, the expression levels of VcARF1-1 and VcARF2-1 at pH 5.2, 5.8, 6.8 were 47.2%-50.8% and 39.5%-82.3% of the ones at pH 4.8, respectively. In contrast, an enhanced expression was observed only at pH 5.2 and 5.8 for VcARF7-1 and VcARF9-1, respectively (Fig. 8D). No significant change was observed for VcARF4-1. When compared with their publicly available transcript profiling, 5 out of 6 genes (VcARF1-1, VcARF2-5, VcARF4-1, VcARF8-4, VcARF18-1) showed similar tendency between expression pattern and transcript profiling in V. corymbosum (Fig. 8A and 8C-8D). These observations suggested that VcARF family, at least a subset of VcARF genes, might be involved in the response to pH stress.

Discussion

Plant ARF genes act as key components of auxin signaling pathway to mediate the expression of auxin-responsive genes [11, 12]. In the present study, 60 VcARF genes were identified in blueberry, and the number of VcARF family members is bigger than the ones in apple (28), grape (26), Arabidopsis (23), sweet orange (19), and poplar (21) [23,24,25,26, 37]. The genome size (haploid) of blueberry is approximately 600 Mb [47], and the annotation of this genome has provided a nonredundant set of 56,087 genes [45], which is roughly 2.05 times as many genes as were annotated in Arabidopsis (27,411, TAIR10). Thus, it is reasonable for blueberry to have a large number of ARF genes. Phylogenetic analysis indicated that VcARFs can be clustered together with the ARFs from other plant species (Fig. 2), suggesting they might have undergone similar evolutionary diversification. Noticeably, 9 digenic, 5 trigenic and 6 tetragenic VcARF pairs showed very high identity (more than 95% at nucleotide level) to each other (Fig. 1 and Table S2), implying that they might have restricted functional diversification. Gene family generally arises from gene duplication during evolution, therefore leading to the acquisition of neofunctionalizations and the emergence of backup or redundant genes [48]. It was estimated that at least three rounds of whole-genome duplications occurred during the evolution of blueberry species [9], which is supposed to facilitate the generation of multiple copy genes. Thus, we assumed that the duplication events of chromosomal segment and whole genome are primarily responsible for the expansion of ARF genes in blueberry.

Considerable evidences indicated that ARFs play important roles during fruit growth as key components of auxin signaling pathway. For example, SlARF10 and SlARF6A facilitate chlorophyll accumulation through activating the expression of SlGLK1 [49, 50], while MdARF106 in 'Royal Gala' apples is expressed during cell division and expansion, suggesting a potential role in the regulation of fruit size [51]. Previously, we revealed that VcARF18 exhibited high transcript accumulation at fruit early developmental stages and remarkably declined at the initiation of fruit maturation [3]. In the present study, 29 VcARFs showed relatively high expression at the pad stage, and gradually decreased as fruit grows and ripens (Fig. 7). These observations suggested that VcARFs possibly exert functions during fruit growth. Fruit maturation can be accompanied by a series of cellular, molecular, biochemical, and structural changes [42]. It has been documented that ARF genes are involved in the regulation of fruit maturation. For example, MdARF5 activates the expression of ethylene-related genes (MdERF2, MdACSs, MdACOs) to initiate fruit maturation in apple [52]; CpARF2 is implicated in the mediation of papaya fruit maturation by promoting the transcriptional activity of CpEIL1 (a key component of ethylene signaling) and the stabilization of CpEIL1 protein [53]; MdARF13 can negatively regulate anthocyanin accumulation of apple fruit directly through repressing the expression of MdDFR [54]. In the present study, 9 VcARFs showed high transcript abundance at maturation stage as compared to early developmental stages, implying a potential role during fruit maturation. Intriguingly, the previous data provided by Gupta et al. [45] indicated that the expressions of three genes (VcACO, CUFF.8159.1, VcEIL1, CUFF.53576; VcDFR, CUFF.38755) were up-regulated as fruit develops and ripens, whereas the chlorophyll-related gene VcGLK1 was transcriptionally down-regulated (Fig S2). These results implied that co-expression pattern might exist between VcARFs and their targets or downstream genes. Thus, the transcript profiles of VcARFs supported that ARF genes may exert important functions in the regulation of fruit growth and maturation.

Blueberry prefers to grow in acidic soils, and neutral to basic pH is generally stressful to the commonly cultivated blueberry species [36]. ARFs are considered as key regulators to respond to various stresses [11, 12]. However, it is unclear if ARFs perform functions in plant response to pH stress. In the present study, similar accumulation patterns were observed for several ARFs (VcARF1s and VcARF18s) in pH-sensitive and pH-tolerant blueberry species (Fig. 8), suggesting some common roles of ARF family in response to pH stress in different species. Noticeably, a group of VcARFs showed different, even opposite, transcriptional response to pH stress between pH-sensitive and pH-tolerant species (Fig. 8), implying that they might contribute to different tolerance capacity to pH stress. Previous studies indicated that plant adaptation to high pH soil requires an effective transcriptional regulation mainly associated with nutrition balance, ROS-mediated responses, detoxification and cell wall [36, 55]. It has been addressed that AtARF2 acts as transcriptional repressor to participate in the regulation of K+ uptake through mediating AtHAK5 transcription in Arabidopsis [56]; OsARF16 is required for iron deficiency response in rice [57]; AtARF3/ETT is a positive player in regulating the activity of pectin methylesterase (PME) in the cell wall of Arabidopsis [58]; loss of function of SlARF4 confers plant tolerance to water deficit by enhancing Superoxide Dismutase (SOD) and antioxidant substances in tomato [41]. It is possible that the transcriptional alteration of VcARFs under different pH conditions might be required for adjusting nutrient availability, ROS-mediated responses, detoxification or/and cell wall. Also, it is worth to explore if VcARFs are involved in the adjustment of the internal pH value. Interestingly, the previous transcriptomic study indicated that all the three genes (VcHAK, CUFF.225.1; VcPME, CUFF.59080.1; VcSOD, CUFF.32906.1) were transcriptionally suppressed in pH-tolerant species under the condition of pH 6.5 (Fig. S2) [59]. Thus, we proposed an assumption that ARF family in blueberry might be involved in response to pH stress possibly via affecting nutrition balance, ROS-mediated responses, detoxification and cell wall.

Conclusions

In conclusion, 60 ARF genes were identified and characterized in blueberry. The combination analysis of gene structures, motif architectures, phylogenetic relationship, sequence identity and miRNA responsive elements suggested their conservation and divergence in features and functional roles across plant species. Furthermore, we assumed that VcARFs might have potential roles during fruit development and in response to pH stress based on their transcript profiles. These findings will contribute to future research for documenting the functional roles of ARFs and their regulatory mechanisms in blueberry, especially fruit development and adaption to pH stress.

Materials and methods

Identification of VcARF family genes

The sequences of publicly known ARF genes in Arabidopsis, apple, grape, tomato and rice were downloaded from NCBI (www.ncbi.nlm.nih.gov), TAIR (www.arabidopsis.org) or RAP-DB (https://rapdb.dna.affrc.go.jp/) database. All the downloaded sequences were applied to BLASTn search against Vaccinium corymbosum cv. Draper genome v1.0 (www.vaccinium.org), and all the potential hits to the conserved regions of ARF genes were subsequently collected. Furthermore, redundant sequences were removed from all the potential hits, and then the complete transcript sequences were retrieved from the database of Vaccinium corymbosum GDV RefTrans v1 (www.vaccinium.org). Each of the predicted VcARF proteins was applied to a confirmation of auxin response factor using the online program Pfam (http://pfam.xfam.org/). The subcellular localizations of VcARF proteins were predicted using the online program WoLF pSORT (https://wolfpsort.hgc.jp/).

Chromosomal locations of VcARFs and phylogenetic analysis of ARF genes

All the 60 VcARF genes were mapped to the genome of Vaccinium corymbosum (Draper), and their location information was obtained accordingly. Subsequently, the information regarding their positions, the correspondingly situated scaffolds, and identity (95%) were imported into the software TBtools to generate a circle plot [60]. The CDS sequences of ARF genes (26 from grape, 28 from apple, 23 from Arabidopsis, 25 from tomato, and 25 from rice) were applied to generate phylogenetic tree with the above 60 VcARFs. Briefly, multiple sequences alignment of all the ARF genes were performed by Clustal X, and phylogenetic tree was constructed by the MEGA7.0 software with maximum likelihood statistical method [61]. Bootstrap values were calculated using 1000 replicates.

Gene structures, conserved motifs and domains of VcARFs

The exon/intron structures of 60 VcARF genes were drawn using GSDS 2.0 (http://gsds.gao-lab.org/) through comparing their genomic sequences and coding sequences. The conserved motifs of VcARF proteins were analyzed using the online program Multiple Expectation Maximization for Motif Elucidation (http://meme-suite.org/tools/meme). The conserved domains were analyzed using the online program Pfam (http://pfam.xfam.org/).

Promoter analysis of VcARF genes

The transcription start sites (TSS) of VcARFs were predicted by the online program TSSP in Softberry (http://linux1.softberry.com/berry.phtml?topic=tssp&group=-programs&subgroup=promoter). Subsequently, a 1,500 bp interval upstream of the TSS was considered as promoter and applied to the online program PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for promoter analysis.

Prediction of miRNA-targeted VcARFs

The online programe psRNATarget (http://plantgrn.noble.org/psRNATarget/home) was used to predict miRNA-targeted VcARFs. Briefly, the sequences of sixty VcARFs and blueberry miRNAs identified previously [3] were submitted to psRNATarget, and the parameters were set as default with a maximum expectation value of 3.0. The diagrams were drawn based on the gene structure accordingly.

Expression profiling of VcARFs during fruit development and in response to pH stress

To understand the expression patterns of VcARF genes in response to pH stress, their publicly available transcript profiling data [59] were retrieved at the database GDV (www.vaccinium.org) regarding blueberry root (two species: pH-sensitive Vaccinium corymbosum and pH-tolerant Vaccinium arboretum) at its preferred pH of 4.5 and near neutral pH 6.5. Briefly, the transcript ID of each ARF gene was obtained using sequence search. Subsequently, the transcript profiling data were retrieved using the online program “Expression Heatmap” (www.vaccinium.org/node/854703). The transcript profiling data of VcARFs during fruit development (five developmental stages such as pads, cups, green, pink, and ripe) were extracted from the previous study reported by Gupta et al. [45]. All the heatmaps were generated using the software TBtools [60].

The pH of the woody plant medium (WPM, PhytoTech, USA) was adjusted using 1 M KOH until a desired final pH 4.8, 5.2, 5.8, or 6.8. Five young tissue culture seedlings with similar growth were transferred to the WPM medium with different pHs, and five Petri plates were used for each treatment. The seedlings with different pH treatment were grown for two weeks under the condition of 16/8 h photoperiod at 20 °C. Subsequently, young leaves of the treated seedlings were collected for RNA extraction.

Total RNAs were extracted from fruit at five developmental stages [green pad (GPS), green cup II (GCS), light green/white (FWS), pink (FPS) and blue (FMS) fruits] and young leaves of tissue culture seedlings of V. corymbosum (cultivar ‘Northland’) under pH 4.8, 5.2, 5.8, 6.8. cDNAs were synthesized using StarScript II First-strand cDNA Synthesis Mix With gDNA Remover Kit (GenStar, China). qRT-PCR was conducted using the Bio-Rad CFX Connect Real-Time PCR Detection System with the reagent of 2 × RealStar Green Fast Mixture (GenStar, China). Data analysis were performed using the software Bio-Rad CFX Manager, and VcACTIN was set as an internal reference for data normalization. Three biological replicates for each sample were conducted with three technical replicates, and error bars indicate SE. Statistical significance of the data was analyzed using ANOVA with LSD test, and p-value < 0.05 was considered to be statistically significant. Primer information is listed in Table S3.

Availability of data and materials

The publicly available transcript profiling data regarding the response to different pH were retrieved at the database GDV (www.vaccinium.org). All data generated or analysed during this study are included in this published article [and its supplementary information files].

References

  1. Routray W, Orsat V. Blueberries and their anthocyanins: factors affecting biosynthesis and properties. Compr Rev Food Sci F. 2011;10(6):303–20.

    Article  CAS  Google Scholar 

  2. Gao X, Walworth AE, Mackie C, Song GQ. Overexpression of blueberry FLOWERING LOCUS T is associated with changes in the expression of phytohormone-related genes in blueberry plants. Hortic Res. 2016;3:16053.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Hou Y, Zhai L, Li X, Xue Y, Wang J, Yang P, Cao C, Li H, Cui Y, Bian S. Comparative analysis of fruit ripening-related miRNAs and their targets in blueberry using small RNA and degradome sequencing. Int J Mol Sci. 2017;18(12):2767.

    Article  PubMed Central  CAS  Google Scholar 

  4. Hou YM, Li HX, Zhai LL, Xie X, Li XY, Bian SM. Identification and functional characterization of the Aux/IAA gene VcIAA27 in blueberry. Plant Signal Behav. 2020;15(1):1700327.

    Article  PubMed  CAS  Google Scholar 

  5. Li X, Hou Y, Xie X, Li H, Li X, Zhu Y, Zhai L, Zhang C, Bian S. A blueberry MIR156a-SPL12 module coordinates the accumulation of chlorophylls and anthocyanins during fruit ripening. J Exp Bot. 2020;71(19):5976–89.

    Article  CAS  PubMed  Google Scholar 

  6. Lin T, Walworth A, Zong X, Danial GH, Tomaszewski EM, Callow P, Han X, Irina Zaharia L, Edger PP, Zhong GY, et al. VcRR2 regulates chilling-mediated flowering through expression of hormone genes in a transgenic blueberry mutant. Hortic Res. 2019;6:96.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Plunkett BJ, Espley RV, Dare AP, Warren BAW, Grierson ERP, Cordiner S, Turner JL, Allan AC, Albert NW, Davies KM, et al. MYBA From blueberry (Vaccinium Section Cyanococcus) is a subgroup 6 type R2R3MYB transcription factor that activates anthocyanin production. Front Plant Sci. 2018;9:1300.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Qi X, Ogden EL, Ehlenfeldt MK, Rowland LJ. Dataset of de novo assembly and functional annotation of the transcriptome of blueberry (Vaccinium spp.). Data Brief. 2019;25:104390.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Wang Y, Nie F, Shahid MQ, Baloch FS. Molecular footprints of selection effects and whole genome duplication (WGD) events in three blueberry species: detected by transcriptome dataset. BMC Plant Biol. 2020;20(1):250.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Zifkin M, Jin A, Ozga JA, Zaharia LI, Schernthaner JP, Gesell A, Abrams SR, Kennedy JA, Constabel CP. Gene expression and metabolite profiling of developing highbush blueberry fruit indicates transcriptional regulation of flavonoid metabolism and activation of abscisic acid metabolism. Plant Physiol. 2012;158(1):200–24.

    Article  CAS  PubMed  Google Scholar 

  11. Lanctot A, Nemhauser JL. It’s Morphin’ time: how multiple signals converge on ARF transcription factors to direct development. Curr Opin Plant Biol. 2020;57:1–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Chandler JW. Auxin response factors. Plant Cell Environ. 2016;39(5):1014–28.

    Article  CAS  PubMed  Google Scholar 

  13. Zhang MM, Zhang HK, Zhai JF, Zhang XS, Sang YL, Cheng ZJ. ARF4 regulates shoot regeneration through coordination with ARF5 and IAA12. Plant Cell Rep. 2021;40(2):315–25.

    Article  CAS  PubMed  Google Scholar 

  14. Wang YC, Wang N, Xu HF, Jiang SH, Fang HC, Su MY, Zhang ZY, Zhang TL, Chen XS. Auxin regulates anthocyanin biosynthesis through the Aux/IAA-ARF signaling pathway in apple. Hortic Res. 2018;5:59.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Wang X, Yu R, Wang J, Lin Z, Han X, Deng Z, Fan L, He H, Deng XW, Chen H. The asymmetric expression of SAUR genes mediated by ARF7/19 promotes the gravitropism and phototropism of plant hypocotyls. Cell Rep. 2020;31(3):107529.

    Article  CAS  PubMed  Google Scholar 

  16. Zhang CL, Wang GL, Zhang YL, Hu X, Zhou LJ, You CX, Li YY, Hao YJ. Apple SUMO E3 ligase MdSIZ1 facilitates SUMOylation of MdARF8 to regulate lateral root formation. New Phytol. 2021;229(4):2206–22.

    Article  CAS  PubMed  Google Scholar 

  17. Lee K, Park OS, Seo PJ. Arabidopsis ATXR2 deposits H3K36me3 at the promoters of LBD genes to facilitate cellular dedifferentiation. Sci Signal. 2017;10(507):eaan0316.

    Article  PubMed  CAS  Google Scholar 

  18. Lee K, Park OS, Seo PJ. JMJ30-mediated demethylation of H3K9me3 drives tissue identity changes to promote callus formation in Arabidopsis. Plant J. 2018;95(6):961–75.

    Article  CAS  PubMed  Google Scholar 

  19. Su Z, Wang N, Hou Z, Li B, Li D, Liu Y, Cai H, Qin Y, Chen X. Regulation of female germline specification via small RNA mobility in Arabidopsis. Plant Cell. 2020;32(9):2842–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhang X, Yan F, Tang Y, Yuan Y, Deng W, Li Z. Auxin response gene SlARF3 plays multiple roles in tomato development and is involved in the formation of epidermal cells and trichomes. Plant Cell Physiol. 2015;56(11):2110–24.

    CAS  PubMed  Google Scholar 

  21. Herud O, Weijers D, Lau S, Jurgens G. Auxin responsiveness of the MONOPTEROS-BODENLOS module in primary root initiation critically depends on the nuclear import kinetics of the Aux/IAA inhibitor BODENLOS. Plant J. 2016;85(2):269–77.

    Article  CAS  PubMed  Google Scholar 

  22. Ulmasov T, Hagen G, Guilfoyle TJ. ARF1, a transcription factor that binds to auxin response elements. Science. 1997;276(5320):1865–8.

    Article  CAS  PubMed  Google Scholar 

  23. Luo XC, Sun MH, Xu RR, Shu HR, Wang JW, Zhang SZ. Genomewide identification and expression analysis of the ARF gene family in apple. J Genet. 2014;93(3):785–97.

    Article  PubMed  Google Scholar 

  24. Wan S, Li W, Zhu Y, Liu Z, Huang W, Zhan J. Genome-wide identification, characterization and expression analysis of the auxin response factor gene family in Vitis vinifera. Plant Cell Rep. 2014;33(8):1365–75.

    Article  CAS  PubMed  Google Scholar 

  25. Li SB, OuYang WZ, Hou XJ, Xie LL, Hu CG, Zhang JZ. Genome-wide identification, isolation and expression analysis of auxin response factor (ARF) gene family in sweet orange (Citrus sinensis). Front Plant Sci. 2015;6:119.

    PubMed  PubMed Central  Google Scholar 

  26. Kalluri UC, Difazio SP, Brunner AM, Tuskan GA. Genome-wide analysis of Aux/IAA and ARF gene families in Populus trichocarpa. BMC Plant Biol. 2007;7:59.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Tang Y, Bao X, Liu K, Wang J, Zhang J, Feng Y, Wang Y, Lin L, Feng J, Li C. Genome-wide identification and expression profiling of the auxin response factor (ARF) gene family in physic nut. PLoS ONE. 2018;13(8):e0201024.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Pei Q, Li N, Yang Q, Wu T, Feng S, Feng X, Jing Z, Zhou R, Gong K, Yu T, et al. Genome-wide identification and comparative analysis of ARF family genes in three Apiaceae species. Front Genet. 2020;11:590535.

    Article  PubMed  CAS  Google Scholar 

  29. Wu J, Wang F, Cheng L, Kong F, Peng Z, Liu S, Yu X, Lu G. Identification, isolation and expression analysis of auxin response factor (ARF) genes in Solanum lycopersicum. Plant Cell Rep. 2011;30(11):2059–73.

    Article  CAS  PubMed  Google Scholar 

  30. Wang D, Pei K, Fu Y, Sun Z, Li S, Liu H, Tang K, Han B, Tao Y. Genome-wide analysis of the auxin response factors (ARF) gene family in rice (Oryza sativa). Gene. 2007;394(1–2):13–24.

    Article  CAS  PubMed  Google Scholar 

  31. Bargmann BO, Vanneste S, Krouk G, Nawy T, Efroni I, Shani E, Choe G, Friml J, Bergmann DC, Estelle M, et al. A map of cell type-specific auxin responses. Mol Syst Biol. 2013;9:688.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Roosjen M, Paque S, Weijers D. Auxin response factors: output control in auxin biology. J Exp Bot. 2018;69(2):179–88.

    Article  CAS  PubMed  Google Scholar 

  33. Ulmasov T, Hagen G, Guilfoyle TJ. Activation and repression of transcription by auxin-response factors. Proc Natl Acad Sci U S A. 1999;96(10):5844–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kato H, Ishizaki K, Kouno M, Shirakawa M, Bowman JL, Nishihama R, Kohchi T. Auxin-mediated transcriptional system with a minimal set of components is critical for morphogenesis through the life cycle in Marchantia polymorpha. PLoS Genet. 2015;11(5):e1005084.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Galli M, Khakhar A, Lu Z, Chen Z, Sen S, Joshi T, Nemhauser JL, Schmitz RJ, Gallavotti A. The DNA binding landscape of the maize AUXIN RESPONSE FACTOR family. Nat Commun. 2018;9(1):4526.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Paya-Milans M, Nunez GH, Olmstead JW, Rinehart TA, Staton M. Regulation of gene expression in roots of the pH-sensitive Vaccinium corymbosum and the pH-tolerant Vaccinium arboreum in response to near neutral pH stress using RNA-Seq. BMC Genomics. 2017;18:580.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Okushima Y, Overvoorde PJ, Arima K, Alonso JM, Chan A, Chang C, Ecker JR, Hughes B, Lui A, Nguyen D, et al. Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: Unique and overlapping functions of ARF7 and ARF19. Plant Cell. 2005;17(2):444–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Xu L, Wang D, Liu S, Fang Z, Su S, Guo C, Zhao C, Tang Y. Comprehensive atlas of wheat (Triticum aestivum L.) AUXIN RESPONSE FACTOR expression during male reproductive development and abiotic stress. Front Plant Sci. 2020;11:586144.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Nan Q, Qian D, Niu Y, He Y, Tong S, Niu Z, Ma J, Yang Y, An L, Wan D, et al. Plant actin-depolymerizing factors possess opposing biochemical properties arising from key amino acid changes throughout evolution. Plant Cell. 2017;29(2):395–408.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Liu S, Zhang Y, Feng Q, Qin L, Pan C, Lamin-Samu AT, Lu G. Tomato AUXIN RESPONSE FACTOR 5 regulates fruit set and development via the mediation of auxin and gibberellin signaling. Sci Rep. 2018;8(1):2971.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Chen M, Zhu X, Liu X, Wu C, Yu C, Hu G, Chen L, Chen R, Bouzayen M, Zouine M, et al. Knockout of Auxin Response Factor SlARF4 Improves Tomato Resistance to Water Deficit. Int J Mol Sci. 2021;22(7):3347.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kumar R, Tyagi AK, Sharma AK. Genome-wide analysis of auxin response factor (ARF) gene family from tomato and analysis of their role in flower and fruit development. Mol Genet Genomics. 2011;285(3):245–60.

    Article  CAS  PubMed  Google Scholar 

  43. Dai X, Lu Q, Wang J, Wang L, Xiang F, Liu Z. MiR160 and its target genes ARF10, ARF16 and ARF17 modulate hypocotyl elongation in a light, BRZ, or PAC-dependent manner in Arabidopsis: miR160 promotes hypocotyl elongation. Plant Sci. 2021;303:110686.

    Article  CAS  PubMed  Google Scholar 

  44. Yao X, Chen J, Zhou J, Yu H, Ge C, Zhang M, Gao X, Dai X, Yang ZN, Zhao Y. An essential role for miRNA167 in maternal control of embryonic and seed development. Plant Physiol. 2019;180(1):453–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Gupta V, Estrada AD, Blakley I, Reid R, Patel K, Meyer MD, Andersen SU, Brown AF, Lila MA, Loraine AE. RNA-Seq analysis and annotation of a draft blueberry genome assembly identifies candidate genes involved in fruit ripening, biosynthesis of bioactive compounds, and stage-specific alternative splicing. Gigascience. 2015;4:5.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Perez-Martin L, Busoms S, Tolra R, Poschenrieder C. Transcriptomics reveals fast changes in salicylate and jasmonate signaling pathways in shoots of carbonate-tolerant Arabidopsis thaliana under bicarbonate exposure. Int J Mol Sci. 2021;22(3):1226.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Costich DE, Ortiz R, Meagher TR, Bruederle LP, Vorsa N. Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry. Theor Appl Genet. 1993;86(8):1001–6.

    Article  CAS  PubMed  Google Scholar 

  48. Cannon SB, Mitra A, Baumgarten A, Young ND, May G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 2004;4:10.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Yuan Y, Mei L, Wu M, Wei W, Shan W, Gong Z, Zhang Q, Yang F, Yan F, Zhang Q, et al. SlARF10, an auxin response factor, is involved in chlorophyll and sugar accumulation during tomato fruit development. J Exp Bot. 2018;69(22):5507–18.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Yuan Y, Xu X, Gong Z, Tang Y, Wu M, Yan F, Zhang X, Zhang Q, Yang F, Hu X, et al. Auxin response factor 6A regulates photosynthesis, sugar accumulation, and fruit development in tomato. Hortic Res-England. 2019;6:85.

    Article  CAS  Google Scholar 

  51. Devoghalaere F, Doucen T, Guitton B, Keeling J, Payne W, Ling TJ, Ross JJ, Hallett IC, Gunaseelan K, Dayatilake GA, et al. A genomics approach to understanding the role of auxin in apple (Malus x domestica) fruit size control. BMC Plant Biol. 2012;12:7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Yue P, Lu Q, Liu Z, Lv T, Li X, Bu H, Liu W, Xu Y, Yuan H, Wang A. Auxin-activated MdARF5 induces the expression of ethylene biosynthetic genes to initiate apple fruit ripening. New Phytol. 2020;226(6):1781–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Zhang T, Li W, Xie R, Xu L, Zhou Y, Li H, Yuan C, Zheng X, Xiao L, Liu K. CpARF2 and CpEIL1 interact to mediate auxin-ethylene interaction and regulate fruit ripening in papaya. Plant J. 2020;103(4):1318–37.

    Article  CAS  PubMed  Google Scholar 

  54. Wang YC, Wang N, Xu HF, Jiang SH, Fang HC, Su MY, Zhang ZY, Zhang TL, Chen XS. Auxin regulates anthocyanin biosynthesis through the Aux/IAA-ARF signaling pathway in apple. Hortic Res-England. 2018;5:59.

    Article  CAS  Google Scholar 

  55. Shavrukov Y, Hirai Y. Good and bad protons: genetic aspects of acidity stress responses in plants. J Exp Bot. 2016;67(1):15–30.

    Article  CAS  PubMed  Google Scholar 

  56. Zhao S, Zhang M-L, Ma T-L, Wang Y. Phosphorylation of ARF2 relieves its repression of transcription of the K+ transporter gene HAK5 in response to low potassium stress. Plant Cell. 2016;28(12):3005–19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Shen C, Yue R, Sun T, Zhang L, Yang Y, Wang H. OsARF16, a transcription factor regulating auxin redistribution, is required for iron deficiency response in rice (Oryza sativa L.). Plant science. 2015;231:148–58.

    Article  CAS  PubMed  Google Scholar 

  58. Andres-Robin A, Reymond MC, Dupire A, Battu V, Dubrulle N, Mouille G, Lefebvre V, Pelloux J, Boudaoud A, Traas J, et al. Evidence for the regulation of gynoecium morphogenesis by ETTIN via cell wall dynamics. Plant Physiol. 2018;178(3):1222–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Paya-Milans M, Nunez GH, Olmstead JW, Rinehart TA, Staton M. Regulation of gene expression in roots of the pH-sensitive Vaccinium corymbosum and the pH-tolerant Vaccinium arboreum in response to near neutral pH stress using RNA-Seq. BMC Genomics. 2017;18(1):580.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R. TBtools: An Integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020;13(8):1194–202.

    Article  CAS  PubMed  Google Scholar 

  61. Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis Version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This work was supported by the National Natural Science Foundation of China [Grant number 31872075, 2019–2022].

Author information

Author notes

  1. Xuyan Li, Xiaoyi Zhang and Tianran Shi contributed equally to this work.

Authors and Affiliations

  1. College of Plant Science, Jilin University, Changchun, China

    Xuyan Li, Xiaoyi Zhang, Tianran Shi, Min Chen, Chengguo Jia, Jingying Wang, Junyou Han & Shaomin Bian

  2. Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Research & Development Center of Blueberry, Beijing, 100083, China

    Zhixia Hou

Authors
  1. Xuyan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaoyi Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tianran Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Min Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chengguo Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jingying Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhixia Hou
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Junyou Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Shaomin Bian
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.B. and X.L. designed the experiments; X.L., X.Z, T.S., M.C, Z.H., J.H. and C.J. performed data analyses; X.L., T. S. and J.W. performed the experiments; S.B. and J.H. wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Junyou Han or Shaomin Bian.

Ethics declarations

Ethics approval and consent to participate

No specific permit is required for the samples in this study. We comply with relevant institutional, national, and international guidelines and legislation for plant studies.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1:

 Table S1. Characterization of the ARF gene familyin blueberry.

Additional file 2:

Table S2. Identities of VcARF gene pairs and their corresponding Ka/Ks values.

Additional file 3:

Table S3. Primers used in the study

Additional file 4:

 Figure S1. Phylogenetic analysis of ARF genes in blueberry and Arabidopsis. The CDS sequences of AtARFs were downloaded from the TAIR website (www.arabidopsis.org). A maximum likelihood tree was generated with the CDS sequences of the ARF genes using the MEGA7 software. 

Additional file 5:

Figure S2.  Transcript profiling of the potential targets or downstream genes of VcARFs. (A) Transcript profiling during fruit development (five stages: pad, cup, mg, pink, ripe). (B-C) Transcript profiling in response to different pH conditions (pH4.5 and pH6.5) in pH-sensitive Vaccinium corymbosum  (B) and pH-tolerant Vaccinium arboretum (C). The color scale beside the heat map indicates gene expression levels, low transcript abundance indicated by blue color and high transcript abundance indicated by red color. The heatmaps were generated using the software TBtools (Version 1.098689, https://github.com/CJ-Chen/TBtools/releases). 

Additional file 6.

 Raw data for qRT-PCR investigation during fruit development.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, X., Zhang, X., Shi, T. et al. Identification of ARF family in blueberry and its potential involvement of fruit development and pH stress response. BMC Genomics 23, 329 (2022). https://doi.org/10.1186/s12864-022-08556-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12864-022-08556-y

Keywords

  • Blueberry
  • ARF family
  • Gene structure
  • Domain and motif compositions
  • Expression pattern
  • Fruit development
  • Response to pH stress

BMC Genomics

ISSN: 1471-2164

Contact us