Genome-wide identification and expression analysis of ethylene responsive factor family transcription factors in Juglans regia

Introduction

Juglans regia is an economic tree species and is distributed widely all over the world (Abdallah et al., 2015). In China, walnut has become an important woody oil tree species for Poverty Alleviation and Rural Revitalization (Yang et al., 2019). However, like other plants, the growth and development of walnut is restricted by biotic (such as pests, diseases) and abiotic factors (such as moisture, temperature, and light). These factors cause a sharp reduction in the yield and quality of walnut. When exposed to external stimuli, plants mobilise protective mechanisms to reduce damage through many pathways, including releasing stress signals, adapting to stress stimuli, activating a series of molecular pathways, regulating related gene expression, and physiological responses (Hilker & Schmülling, 2019). For example, WRKY, NAC, MYB, AP2/ERF, and bZIP transcription factor (TF) gene families in Arabidopsis thaliana were highly enriched and involved in regulating the expression of 56% of common genes in response to drought and cold stresses (Sharma et al., 2018). GmWRKY54 transgenic soybean improved drought tolerance through abscisic acid (ABA) and Ca²⁺ signalling pathways (Wei et al., 2019). To enhance tolerance to high temperature stress, the expression of walnut JrGRAS2 stimulates the transcription activity of heat shock proteins (Yang et al., 2018). Therefore, the identification of important TF families can reveal the stress adaptation mechanism of walnut. It will provide a theoretical basis for adversity-related molecular breeding.

TFs are the key molecules for the regulation of gene expression and exist in all organisms. APETALA2/ethylene responsive factor (AP2/ERF) is one of the TF families in plants. After the first AP2/ERF family TF was isolated and identified from A. thaliana in 1994 (Jofuku et al., 1994), it had been reported widely in various plants, such as Ananas comosus (Zhang et al., 2021), Betula platyphylla (Lv et al., 2020), and Raphanus sativus (Karanja et al., 2019). According to the type and number of AP2/ERF conserved domain, AP2/ERF TFs can be divided into the following subfamilies: APETALA2 (AP2), related to ABI3/VP1 (RAV), dehydration-responsive element binding protein (DREB), ERF, and Soloist (Sakuma et al., 2002). AP2 subfamily, containing two highly conservative AP2/ERF domains, is involved in cell growth and differentiation (Luo et al., 2020). RAV subfamily, including one AP2/ERF and one B3 domains, is involved in plant flowering and stress response (Zhao et al., 2017). DREB subfamily has an AP2/ERF domain, whose amino acid 14 and 19 are valine and glutamic acid, respectively. DREB has been found to be positive response genes in low temperature, drought, and ABA signalling pathways (Sarkar et al., 2019). The ERF subfamily has only one AP2/ERF domain, whose amino acid 14 and 19 are alanine and aspartic acid, respectively, which are the sites that distinguish the subfamily of ERF from DREB. ERF subfamily relates with various stimuli, such as hormones, low temperature, drought, salt, and pathogens (Lv et al., 2020). Soloist is an orphan of the AP2/ERF family with only one AP2/ERF domain, whose amino acid motif and gene structure are significantly different from those of other AP2/ERF subfamilies. Soloist is mainly involved in the response to low temperature and the associated signal transduction pathways (Wang et al., 2018).

Among the subfamilies of AP2/ERF, ERF has received the greatest attention because of its broad response to various stresses. For instance, plants with Arabidopsis ERF96 overexpression displayed enhanced resistance to necrotrophic pathogens, which included the fungus Botrytis cinerea and the bacterium Pectobacterium carotovorum (Fröschel et al., 2019). Ectopic expression of Phaseolus vulgaris ERF35 in tobacco promoted salt stress tolerance (Kavas et al., 2020). Apple ERF38 played positive role in drought tolerance relating to anthocyanin biosynthesis (An et al., 2020). Solanum lycopersicum ERFB3 could respond to cold, heat, and flooding and plays a role in the layout of stress symptoms under cold stress (Klay et al., 2014). Especially in the regulation of drought tolerance, ERF has attracted much attention. For example, the overexpression of barley HvSHN1 in transgenic tobacco improved drought tolerance without compromising growth (Djemal & Khoudi, 2021). In GT31, a sugarcane variety with no tolerance to drought, Saccharum spontaneum SsDREB1L showed higher expression level after re-watering, indicating that SsDREB1L may facilitate plant recovery from drought stress (Li et al., 2021). Tobacco ERF172 improves plant drought tolerance partly by regulating CAT-mediated H₂O₂ homeostasis (Zhao et al., 2020). However, in woody plants, there are little or no reports on the identification of ERF gene family, especially in walnut, where there is no report of ERF in stress response. Therefore, in this study, the walnut ERF TFs were identified and the basic bioinformation, conserved motifs, and evolutionary relationship were analysed. Meanwhile, considering that ERFs are involved in the ethylene (ETH) signalling pathway (Gu et al., 2017; Kazan, 2015; Xie et al., 2019), three stresses (PEG₆₀₀₀, ethephon, and PEG₆₀₀₀+ethephon) were used to assess the potential transcription activity of the selected JrERFs. The results of this study will provide profound platform for subsequent investigation of JrERFs response to stress.

Materials & methods

Results

Sequence characteristics and chromosomal locations of JrERFs

In summary, a total of 44 JrERFs were identified from walnut transcriptome. The ORFs of JrERFs were between 450 bp (JrERF42) and 1,239 bp (JrERF41), consisting 149–412 amino acids. The molecular weight of the proteins ranged from 16.81 kDa (JrERF42) to 43.71 kDa (JrERF41), and the pI ranged from 4.80 (JrERF11) to 9.89 (JrERF03) (Table 1).

These 44 JrERFs were unevenly located on 13 chromosomes (chr1S, chr1D, chr2S, chr2D, chr3S, chr3D, chr4S, chr4D, chr5D, chr6S, chr6D, chr8S, chr8D) of J. regia. The chromosomes 1D, 2S, and 2D covered the most number of JrERFs (6, 7, and 7 genes, respectively), while the chromosomes 1S, 4S and 8D included the least number of JrERFs (1 gene, accordingly). The chromosomes 5S, 7S, and 7D contained no JrERFs (Fig. 1).

The intron and exon structure of JrERFs were analysed by comparing the genomic DNA sequence of JrERFs. The results showed that the gene structures of nine JrERFs (JrERF01, JrERF03, JrERF05, JrERF15, JrERF20, JrERF25, JrERF28, JrERF30, JrERF41) were destroyed by introns, and the other 35 JrERFs contained no intron and their structures are relatively stable (Fig. 2).

The conserved domains of JrERFs

Conserved domain analysis showed that there was a common AP2 conserved domain with different degrees of insertion or deletion in the JrERF proteins (Fig. 3A; Fig. S1). MEME and Tbtools (Chen et al., 2020) were used to construct the conserved domain sequence and sequence logo, and the result suggested that most JrERFs contain conserved motifs. Three motifs (motif1, motif2, and motif3) existed in most of the sequences. We found that motif1–3 may be part of the AP2 domain; sequence similarity was 100% at sites 5, 10, 11, and 12 in motif1, sites 1, 2, 10, 11, 15, 23, and 29 in motif2, and sites 7 and 9 in motif3 (Fig. 3B).

The conservative motif of JrERFs

We used MEME to analyse the motifs in the 44 JrERFs and downloaded the basic information (width and best possible match sequence) (Table 2). The results showed that each motif contained 9–50 amino acids, and each sequence contains 1–6 motifs. Among 44 amino acid sequences, JrERF43 and JrERF44 had six conserved motifs (motif6, motif15, motif3, motif1, motif19, motif9), while JrERF03 had only one (motif2). JrERF12, 17, 32, 36, and 40 had five (motif3, motif1, motif2, motif18, motif5). In addition, the most frequent motifs of JrERFs are motif1 (QRPWGKWAAEIRDPRKKTRVW), motif2 (LGTFDTAEEAARAYDRAALKLRGPKAKLNF), and motif3 (KEKKYRGVR). They represented the AP2 domain and are widely present in the 44 JrERFs (Fig. 4).

Table 2:

Motif sequences identified by MEME tool.

motif	Width	Best possible match
motif1	21	QRPWGKWAAEIRDPRKKTRVW
motif2	30	LGTFDTAEEAARAYDRAALKLRGPKAKLNF
motif3	9	KEKKYRGVR
motif4	29	PELVESLPRPASSSPRDIQAAAAKAAAMK
motif5	13	RRPLPFDLNLPPP
motif6	29	KMRVVRIIVSDPYATDSSSSEDDSEKCVK
motif7	50	TPMFSEGFSSQNQMGFEQPGPJGLNQLTPSQILQIQAQIQLQKQNQQRQQ
motif8	50	KLRKCCKDPYPSLTCLRLDAENSHIGVWQKRAGQRSDSNWIMRIPLGKKN
motif9	38	FFDDFEVCGTEDGGGNELPDWDFADICDDFGWMNEPLN
motif10	50	LVITPPVLSGGAGACELFGWRPKPECFSGAGNPPPVRSEYKGYKMENVDV
motif11	14	MSIMVSALTHVVSG
motif12	18	DYKPLHSSVDAKLQAICQ
motif13	11	MCGGAIISDFI
motif14	20	MPNLLVDMAEGMLVSPPRIN
motif15	50	KRLVREIHJPLVKQPPPKLLQSESSCQDSNNGGRTPKVIEAEKKRVLAKT
motif16	21	MDEEERIALQMIEELLNWNCP
motif17	13	CPVCNIDGCLGCN
motif18	14	GCHSDSDSSSVVDD
motif19	36	LGTFNTPEEASEAYZKKRLEFEAAMAANANSEKSKN

DOI: 10.7717/peerj.12429/table-2

The evolutionary relationship of JrERFs

To analyse the evolutionary relationships of JrERFs, 44 JrERFs, 63 A. thaliana ERFs, and 37 P. trichocarpa ERFs were aligned for the construction of the phylogenetic tree. The results showed that JrERFs can be classified into six groups (B1–B6). Group B6 contained the highest number of JrERF proteins (17 members); groups B2, B3, and B5 had only four members they respectively include JrERF01, JrERF05, JrERF28, JrERF29; JrERF03, JrERF06, JrERF27, JrERF38; JrERF16, JrERF26, JrERF33, JrERF34; groups B4 contained six members (JrERF02, JrERF15, JrERF24, JrERF25, JrERF37, JrERF41); and the remaining nine JrERF proteins were in group B1 (Fig. 5).

Expression of JrERFs in response to drought stress

To explore the potential function of JrERFs in response to drought stress, the expression of all the JrERFs were analysed under PEG₆₀₀₀, ETH, and PEG₆₀₀₀+ETH treatments (Figs. 6–8).

Discussion

Walnut is an important economic species, and like other plants, its growth and development are restricted by adverse environmental conditions (Zhang et al., 2020b). There are little or no reports on ERF, a big subfamily of AP2/ERF playing important roles in stress response, in walnut tree. To increase the yield and quality of walnuts as well as ensure farmers’ economic income and stable development of walnut industry under adverse conditions, it is necessary to elucidate the molecular mechanism associated with adversity adaptation in walnut. Therefore, in this study, a total of 44 JrERFs that may have potential functions in drought stress response were obtained from walnut transcriptome and divided into six groups (Fig. 5). This classification was consistent with previous evolutionary analyses of Apium graveolens (Li et al., 2019a), Zay mays (Hao et al., 2020), and Dimocarpus longan (Zhang et al., 2020a). The various characteristics including ORF length, pI, amino acid number, and molecular weight of the walnut ERF family had a large span (Table 1). The phenomenon is similar to the sequence characteristics of Arabidopsis and P. trichocarpa (Zhuang et al., 2008), indicating that the ERF family of walnut has a certain similarity in sequence characteristics with other species.

Multiple alignment result showed that the 44 JrERFs are highly conserved with AP2 domain (Fig. 3A; Fig. S1), and this was consistent with the conserved region of AtERFs (Fig. S2). This result further confirmed that ERF TFs have highly conserved structures in procession of species evolutionary (Nakano et al., 2006). Gene structure analysis showed that 79.55 % of the JrERFs had no intron, implying that most JrERFs have relatively stable gene structure (Fig. 2), which was similar to that observed with AP2/ERF genes from tartary buckwheat (Fagopyum tataricum) (Liu et al., 2019). Most JrERFs contained motif1, motif2, and motif3, which were related to AP2 domain (Fig. 3B). Motif1–motif19 were distributed in the 44 JrERFs at different degrees (Fig. 4), which was also similar to what was observed with ERF TFs from F. tataricum and Medicago sativa (Jin et al., 2019; Liu et al., 2019). Because different motifs and the number of motifs are related to functions (Tripathi et al., 2020), our results suggest that JrERFs may play different roles in plant stress response or other biological functions.

Considering that drought has a great impact on the walnut industry (Knipfer et al., 2018), the responses of the identified 44 JrERFs to PEG₆₀₀₀ were analysed in order to uncover the functions of walnut AP2/ERF family in response to drought stress. The results showed that most of the JrERFs were induced by PEG₆₀₀₀ (Fig. 6). The expression of genes in response to different stresses can often effectively predict their potential functions. For example, it has been reported that the expression levels of various AP2/ERF TFs (Bra-ERF036, Bra-ERF069a, and Bra-ERF104a) from Brassica oleracea are rapidly up-regulated by drought stress, which confirms their important roles in tolerance to abiotic stress (Li et al., 2017). IbRAP2-12, a member of sweet potato ERF family, was rapidly enhanced by drought stress and played a crucial role in enhancing plant tolerance to drought stress (Li et al., 2019b). The expression of JrWRKY2 and JrWRKY7 from walnut was enhanced by PEG₆₀₀₀ treatment, and both were further confirmed to be positive TFs in response to drought stress (Yang et al., 2017). According to these findings, we speculate that JrERFs can respond to drought stress to varying degrees and some members may play positive roles in the regulation of drought tolerance.

Ethylene, a plant growth regulator, participates in multiple plant stress response, such as drought, low temperature, salinity, and mechanical damage (Bleecker & Kende, 2000; Johnson & Ecker, 1998; Mizoi, Shinozaki & Yamaguchi-Shinozaki, 2012). In drought response, ethylene plays a positive role in enhancing drought resistance (Wan et al., 2011). ERFs are associated with ethylene response, and most ERFs can be regulated by ethylene (Müller & Munné-Bosch, 2015; Xie et al., 2019). For instance, the OsDERF1 from rice can be activated by ethylene to improve tolerance to drought stress (Wan et al., 2011). Therefore, in order to verify that the response of JrERFs under drought is related to the ethylene signal pathway, walnuts were treated with ETH and PEG₆₀₀₀+ETH, and the expression levels of JrERFs were analysed. Furthermore, the expression levels of JrERFs under the two treatments were compared. The results showed that most JrERFs can be up-regulated by ETH (Fig. 7), suggesting that ETH has regulatory effect on the expression of JrERFs. Moreover, under PEG₆₀₀₀+ETH, the expression levels of 44 JrERFs were obviously higher than those observed under PEG₆₀₀₀ alone (Fig. 8). These results are similar to other reports. For instance, the expression level of ZmEREB180 was significantly improved with increase in the level of ethylene, which benefited the waterlogging tolerance of plant, indicating that the drought response role of ZmEREB180 was mediated by ethylene (Yu et al., 2019). Arabidopsis RAP2.2 regulated the ability of plants to resist hypoxia through ethylene-controlled signal transduction pathways (Hinz et al., 2010). In soybean, ethylene treatment significantly enhanced the expression of GmERF3, and the overexpression of GmERF3 in tobacco improved the salt and drought tolerance of transgenic lines (Zhang et al., 2009). Therefore, we can conclude that the ethylene signalling pathway is involved in the response of JrERFs to drought stress.

In addition, among the 44 JrERFs, JrERF11 showed the highest expression. The expression of JrERF11 reached the peak at 24 h (5.97) under PEG₆₀₀₀+ETH stress, which was 1.41-fold higher than that of other JrERFs at 24 h. JrERF11 contains no intron and is evolutionarily close to PbERF027 from Pyrus brestschneideri (Fig. S3). PbERF027 is involved in the regulation of genes related to plant hormones (Pei et al., 2016). Moreover, the expression of NnERF026, another homologue from Nelumbo nucifera (Fig. S3), could be induced by drought stress and may function positively in drought stress (Cao et al., 2021). Based on these findings, it can be speculated that JrERF11 is a potential useful gene for drought tolerance and its function should be further investigated.

Conclusions

In this study, a total of 44 JrERFs were identified from J. regia and their basic biological information, chromosome locations, gene structure, conserved motifs, phylogenetic relationship were analysed. We found that the JrERFs were highly conserved and belonged to six groups. More than 40% of the JrERFs can be induced by drought stress in the presence of ETH, implying that JrERFs can respond to drought stress through the ethylene signalling pathway. JrERF11 is the most prominently expressed gene and worthy of further study. The result of our study can provide solid foundation for further investigation of JrERFs under multiple abiotic stresses and for exploring the molecular mechanism underlying abiotic stress responses in walnut and other woody plants.

Supplemental Information