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Article

Ethylene Signaling Pathway Genes in Strawberry and Their Expression Patterns during Fruit Ripening

by
Yunting Zhang
1,†,
Meiyi Deng
1,†,
Xianjie Gu
2,
Chenhui Guo
1,
Yan Chen
1,
Yuanxiu Lin
1,
Qing Chen
1,
Yan Wang
1,
Yong Zhang
1,
Ya Luo
1,
Xiaorong Wang
1 and
Haoru Tang
1,*
1
College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China
2
Institute of Pomology & Olericulture, Mianyang Academy of Agricultural Sciences, Mianyang 621000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(7), 1930; https://doi.org/10.3390/agronomy13071930
Submission received: 26 June 2023 / Revised: 19 July 2023 / Accepted: 19 July 2023 / Published: 21 July 2023
(This article belongs to the Special Issue Progress in Horticultural Crops - from Genotype to Phenotype)
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Abstract

:
Ethylene at least partly regulates some aspects during non-climacteric ripening in strawberry. However, the ethylene signaling pathway genes in the strawberry fruit have not been comprehensively and systematically analyzed. In the present study, 15 FaETRs and 14 FaEIN3/EINs were identified in the octoploid strawberry genome. Subcellular localization analysis predicted that FaETRs and FaEIN3/EINs are respectively localized to the endoplasmic reticulum and the nucleus. The phylogenetic trees showed that FaETRs were classified into two subgroups, while FaEIN3/EINs were divided into three clades, which was supported by gene structure and conserved motif analysis. FaETRs and FaEIN3/EINs could interact with several components, such as CTR1, RTE1, EIN2 and ERF1B, in the ethylene signaling pathway by protein–protein interaction network analysis. Transcriptomic data showed that FaETRs were mainly expressed at the early stage of fruit development in three strawberry cultivars. Additionally, a couple of FaETRs (FaETR2 and FaETR13) and FaEINs (FaEIN2 and FaEIN7) could be induced by 1 μM ABA and inhibited by 100 μM nordihydroguaiaretic acid (NDGA, an ABA biosynthesis blocker). These findings suggested that the FaETR- and FaEIN3/EIN-mediated ethylene signaling pathway might play a role in strawberry fruit ripening.

1. Introduction

Ethylene, the simplest olefin gas, is the first gaseous molecule shown to function as a hormone. It is biosynthesized by plants and is well-known to control a variety of aspects of plant growth and development, such as cell division and expansion, seed germination, root hair formation, organ senescence, leaf flower abscission and fruit ripening, as well as responses to various abiotic and biotic stresses, such as wounding, salt, heat, chilling, heavy metals, drought, flooding, insect infestation and pathogen invasion [1,2,3,4].
Ethylene biosynthesis starts from methionine, which sequentially converts into S-adenosyl-l-methionine (SAM) and the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) through two-step enzymatic reactions, catalyzed by SAM synthetase (SAMS) and ACC synthase (ACS). ACC is then metabolized to ethylene by ACC oxidase (ACO) [5]. During the past three decades, a combination of genetic and molecular analyses that relies on the triple-response phenotype as a morphological marker has allowed the characterization of a collection of ethylene-response mutants and pivotal components of the ethylene signaling pathway, which has proposed a primarily linear model of ethylene signal transduction that starts with hormone perception and ends in transcriptional regulation [6]. Briefly, once the ethylene molecule is perceived by ethylene receptors (ETR1, ERS1, ETR2, EIN4 and ERS2) at the endoplasmic reticulum (ER) membrane, the receptors signal to the negative regulator CTR1, a Raf-like serine/threonine (Ser/Thr) kinase, preventing the phosphorylation of the positive regulator EIN2 and causing the C-terminal end of EIN2 to translocate to the nucleus, where the EIN2 C-end leads to the stabilization of EIN3/EILs and the initiation of transcriptional responses to ethylene [7,8].
Fruit ripening, an irreversible phenomenon, is accompanied by genetically programmed and highly coordinated biochemical changes in color, aroma, flavor, texture and nutritional characteristics, and this process is regulated by an intricate network of different phytohormones. According to the respiration pattern and ethylene production during fruit ripening, fleshy fruits are broadly segmented into two groups: climacteric such as tomato, peach and banana and non-climacteric such as strawberry, litchi and citrus [9,10]. The phytohormone ethylene is well-known to be predominately involved in climacteric fruit ripening [11]. Therefore, the importance of the ethylene biosynthetic and signal transduction pathways in climacteric fruits has been deeply and extensively studied including the characterization of gene families involving these pathways [12,13,14]. It has been demonstrated that ABA also plays a synergistic role during climacteric fruit ripening [15,16]. The peak value of ABA content occurs prior to ethylene, and ABA was proposed to be an upstream regulator of the ethylene pathway [17]. Exogenous ABA treatment or manipulation of genes related to ABA signaling and the biosynthetic pathway regulates climacteric fruit ripening by affecting ethylene biosynthesis and signal transduction [16,18,19]. Moreover, auxins, jasmonic acid (JA), gibberellin (GA), brassinosteroid (BR), salicylic acid (SA) and melatonin are involved in climacteric fruit ripening through ethylene-related pathways [20]. In non-climacteric fruits, ABA is mainly regarded to participate in the regulation of fruit ripening [21]. However, an increasing body of evidence suggests ethylene contributes at least partly to the control of some aspects of non-climacteric ripening [22,23,24,25], but significant progress has not been achieved in revealing the role of ethylene in this process. It was demonstrated that ethylene played a potential role in grape development and ripening [22]. Li et al. (2016) [26] summarized the effect of 1-Methylcyclopropene (1-MCP), an ethylene antagonist, on the postharvest storage performance of non-climacteric fruits and proposed that 1-MCP application could be a method of inhibiting the color change, retarding senescence processes and reducing physiological disorders in certain non-climacteric fruits. Moreover, ethylene signaling has been found to participate in regulating non-climacteric fruit ripening as well by interacting with the ABA signaling, as elucidated by AREB/ABF-mediated ACS/ACO expression and ERF-mediated NCED expression [20,21]. In addition, ethylene response factors (ERFs) can affect different aspects (color, aroma and flavor) of fruit ripening and senescence in non-climacteric fruits [27,28,29].
Strawberry, a typical example of a non-climacteric fruit, has great economic and nutritional value because of its health-promoting compounds, pleasant flavor and attractive appearance. ABA is the core factor that promotes strawberry ripening [30]. It has been found that ethylene also accelerates some aspects of strawberry ripening such as anthocyanin accumulation, total sugar increment and cell wall degradation, while 1-MCP treatment has the opposite effect [31,32], and some ripening-related genes could be regulated by ethylene [31,33]. Downregulation of the FaSAMS1 or FaCTR1 expression level by the RNAi technique retarded strawberry red-coloring and softening. Accordingly, ethephon application could promote natural strawberry fruit red-coloring and softening and partially rescue anthocyanin accumulation in FaSAMS1 and FaCTR1-RNAi fruits but could not significantly influence firmness [34]. Additionally, a high transcript level of ethylene receptors seems required for strawberry fruit ripening [35]. Our previous study showed that the expression level of FaERFs was associated with strawberry fruit development and ripening and was correspondingly regulated by exogenous ABA treatment [36]. These findings indicate that the ethylene signal transduction pathway plays a role in strawberry fruit ripening. In this study, the FaETR (starting) and FaEIN3/EIN (ending) genes in the ethylene signal transduction pathway were identified, and their transcript profiles were measured during fruit ripening, with the aim to obtain new information on various components of the pathway and gain insight into their effect on fruit ripening.

2. Materials and Methods

2.1. Plant Materials

The octoploid strawberries (‘Xiaobai’ cultivar) were harvested at three different developmental stages according to the fruit peel color and days after full bloom (DAFB) during ripening (big green, 21 DAFB; turn red, 28 DAFB; full red, 35 DAFB) from a local orchard located in Wenjiang County, Sichuan Province, China, on 12 March 2021. The samples were quickly frozen in liquid nitrogen, delivered to the laboratory and stored in −80 °C freezer for further analysis.

2.2. Screening of Ethylene Signaling Pathway Genes in Strawberry

The files of octoploid cultivated strawberry (Fragaria × ananassa genome v1.0.a1) were downloaded from the Genome Database for Rosaceae (GDR) (https://www.rosaceae.org, accessed on 12 August 2022). Two methods (local Blast and HMMER search) were used to screen ethylene signaling pathway genes in strawberry. The amino acid sequences in this pathway from tomato and Arabidopsis thaliana were used as queries for BLASTP analysis against Fragaria × ananassa Camarosa genome v1.0.a1 (https://www.rosaceae.org, accessed on 14 August 2022). The specific hidden Markov Model (HMM) profile (PF04873) of EIN3/EINs was downloaded from the Pfam database (https://pfam.xfam.org, accessed on 16 August 2022) and used as the query to search for FaEIN3/EINs genes. Finally, all proteins were delivered to the Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 20 August 2022) to confirm the reliability of candidates.

2.3. Physicochemical Property and Subcellular Localization Analysis

The basic physicochemical properties of protein size, grand average of hydropathicity (GRAVY), theoretical pI, aliphatic index, molecular weight (MW), instability index and subcellular localization of FaETR and FaEIN3/EINs were analyzed using ExPASy-ProtParam online servers (http://web.expasy.org/protparam/, accessed on 22 August 2022) and ProtComp v.9.0 (http://linux1.softberry.com/berry.phtml?topic=protcomppl&group=programs&subgroup=proloc, accessed on 22 August 2022), respectively.

2.4. Chromosomal Mapping and Phylogenetic Tree Analysis

The information on the chromosomal locations of FaETR and FaEIN3/EIN genes was retrieved from the annotated file of the GDR database and graphically visualized with Tbtools. A phylogenetic tree of FaETR and FaEIN3/EINs was built with MEGA X using neighbor-joining (NJ) algorithm with bootstrap support (1000 replicates), according to the multiple sequence alignment in MUSCLE.

2.5. Gene Structure and Conserved Motif Analysis

The exon–intron organization of FaETR and FaEIN3/EIN genes was analyzed with the online tool (Gene Structure Display Server v.2.0, http://gsds.cbi.pku.edu.cn, accessed on 25 August 2022) via the comparison of the full-length genome sequence and the corresponding coding sequences. Conserved motifs were characterized using the Multiple Em for Motif Elicitation (MEME) online tool (http://meme-suite.org/tools/meme, accessed on 27 August 2022) with default parameters.

2.6. Protein–Protein Interaction Network Prediction

Fifteen FaETR and fourteen FaEIN3/EIN protein sequences were used as queries; protein–protein interaction network was analyzed with the STRING online tool (https://string-db.org/, accessed on 5 September 2022). The orthologous genes of diploid strawberry (Fragaria × vesca) were selected as references. After mapping, the interaction network was constructed using the highest-score gene (bitscore).

2.7. Transcriptome Analysis during Fruit Development and Ripening

The expression patterns of FaETR and FaEIN3/EIN genes in ethylene signaling pathway were calculated using the transcriptomic data that had been published on the NCBI database (PRJNA552213; PRJNA338879) [37,38]. The fruit samples in the transcriptome (PRJNA552213) were harvested from three stages of middle green (MG), initial red (IR) and full red (FR) of three strawberry varieties (‘Benihoppe’, ‘Xiaobai’ and ‘Snow Princess’). The samples in the transcriptome (PRJNA338879) were obtained from ‘Toyonoka’ cultivar that was subjected to 1 μM ABA and 100 μM nordihydroguaiaretic acid (NDGA, an ABA biosynthesis blocker) treatments.

2.8. RNA Extraction, cDNA Synthesis and qRT-PCR

The total RNA from strawberry varieties (‘Xiaobai’) was isolated using the improved CTAB (cetyltrimethylammonium bromide) method [39]. After evaluation of the quantity and quality with NanoDrop ND 2000 spectrophotometer and agarose gel electrophoresis, the total RNA was transcribed into cDNA using the PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara, Japan) according to the manufacturing instruction. qRT-PCR was performed on the CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA) with SYBR Premix (Takara, Japan). The total 10 μL reaction mixture consisted of 1 μL template (1:10 diluted cDNA), 0.4 μL each primer for a final concentration of 0.4 μM, 5 μL SYBR Premix and 3.2 μL of RNase-free water. Reaction procedure was performed using two-step cycling conditions: 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 58 °C for 20 s and 72 °C for 20 s. Melting curve was inserted after the final cycle, ramping from 65 °C to 95 °C (increment 0.5 °C/5 s). The presence of a single and sharp peak demonstrated the specificity of primer amplification. Controls without template were included in each run to check the potential reagent contamination. Gene-specific primers used for qRT-PCR were designed using Primer 3 and are listed in Table S1. The relative gene expression levels were calculated using the 2−ΔΔCT formula [40].

3. Results

3.1. Identification and Characterization of Ethylene Signaling Pathway Genes in Strawberry

To extensively identify FaETRs and FaEIN3/EINs in the ethylene signaling transduction pathway from the strawberry, both HMM and the local BLAST program were used to scan the strawberry genome database (v1.0.a1). The two gene families had 29 putative members in total, including 15 FaETRs and 14 FaEIN3/EINs. Their sequence feature is analyzed in Table 1 and Table 2. The length of FaETR and FaEIN3/EIN proteins ranged from 553 to 774 and 232 to 797 aa with molecular weight varying from 62,261.50 to 87,072.14 and 26,951.89 to 88,258.07 Da and theoretical pI varying from 5.83 to 7.25 and 5.09 to 6.20, respectively. Instability indexes of most FaETRs and all FaEIN3/EINs were above 40 except for ETR4, ETR5, ETR6 and ETR7, indicating that only ETR4-7 were stable proteins. Subcellular localization analysis predicted that all FaETRs and FaEIN3/EINs were respectively localized to the endoplasmic reticulum (ER) and the nucleus.

3.2. Chromosomal Localization and Phylogenetic Tree

To determine the distribution of FaETR and FaEIN3/EIN genes on chromosomes in strawberry, a chromosome map was constructed according to the genome annotation. The 13 FaETR genes were evenly located on 13 chromosomes, with the remaining two genes (FaETR12 and FaETR15) localized on chromosome 6-3. Chromosomes Fvb1-2, Fvb1-3 and Fvb1-4 had two members of FaEIN3/EINs, while the other eight chromosomes only had one member (Figure 1). To unravel the evolutionary history of the FaETR or FaEIN3/EIN gene family from strawberry and to help in their classification, FaETRs and FaEIN3/EINs from different species were used to build an unrooted phylogenetic tree. The phylogenetic trees showed that FaETR families were divided into two clades. Eight FaETRs (FaETR1-8) in strawberry, together with AtERS1 (AT2G40940) and AtETR1 (AT1G66340) in Arabidopsis and SlETR2 (Solyc07g056580), SlETR1 (Solyc12g011330) and SlETR3 (Solyc09g075440) in tomato, belonged to Class 1. Seven FaETRs (FaETR9-15) in strawberry, together with AtETR2 (AT3G23150), AtERS2 (AT1G04310) and AtEIN4 (AT3G04580) in Arabidopsis and SlETR6 (Solyc09g089610), SlETR4 (Solyc06g053710), SlETR7 (Solyc05g055070) and SlETR5 (Solyc11g006180) in tomato, belonged to Class 2. FaEIN3/EIN families were divided into three classes. Class 1 had four FaEIN3/EINs that were clustered with AtEIL3 (AT1G73730), Class 2 had three FaEIN3/EINs that were clustered with AtEIL5 (AT5G65100), AtEIL4 (AT5G10120) and AtEIL2 (AT5G21120), and Class 3 had seven FaEIN3/EINs that were clustered with AtEIN3 (AT3G20770) and ATEIL1 (AT2G27050) (Figure 2).

3.3. Gene Structure and Conserved Motif

To better understand the gene structural characteristics of the ethylene signaling pathway genes, their intron/exon arrangement was analyzed. All FaETRs had at least one intron and up to six exons. FaETR1-8 clustered into Class 1 contained the most exons, while FaETR9-15 clustered into Class 2 only had two exons. Seven FaEIN3/EINs had no intron, and other members presented the break sequences because of the distribution of different size introns. The conserved motifs of FaETR and FaEIN3/EIN proteins were analyzed using the MEME tool. The closely related proteins in the same clade exhibited similar motif compositions. Remarkably, the similarity in exon–intron structure and motif distribution supported the results from the phylogenetic analysis of genes, indicating that their function was both conserved and diversified (Figure 3).

3.4. Protein–Protein Interaction Network Analysis

A protein–protein interaction network was constructed to predict the molecular interaction of FaETRs and FaEIN3/EINs in the ethylene signaling pathway, based on their orthologous genes in diploid strawberry (Fragaria × vesca). It was predicted that FaETRs interacted with CTR1 and RTE1 and FaEIN3/EINs interacted with EIN2, ERF1B, EBF1 and MAPKs. All proteins are important components in the ethylene signaling pathway (Figure 4).

3.5. Expression Profiles during Fruit Ripening

To identify the FaETR and FaEIN3/EIN genes involved in fruit ripening, their expression patterns during the fruit development of three strawberry cultivars were examined. As shown in Figure 5A, most FaETRs had higher expression levels in the cultivar ‘Benihoppe’. Interestingly, FaETRs were mainly expressed at the early stage of fruit development in three cultivars. FaEIN2, FaEIN4, FaEIN7 and FaEIN10 had the highest expression at the IR stage of ‘Benihoppe’. FaEIN3, FaEIN6, FaEIN8 and FaEIN14 exhibited higher expression over fruit development in ‘Snow Princess’ than the other two cultivars. Moreover, FaEIN5 and FaEIN12 had the highest expression at the IR stage of ‘Snow Princess’ (Figure 5B). The transcript levels of FaETR and FaEIN3/EIN genes in the strawberry fruit exposed to ABA and ABA biosynthesis blocker nordihydroguaiaretic acid (NDGA) treatments were profiled. The results showed that only a couple of FaETRs (FaETR2 and FaETR13) and FaEINs (FaEIN2 and FaEIN7) could be induced by ABA and inhibited by NDGA (Figure 5C,D). In addition, qRT-PCR showed that the expression patterns of FaETR2, FaETR13, FaEIN2 and FaEIN7 were consistent with the transcriptome data (Figure 6).

4. Discussion

A growing body of evidence suggests that ethylene affects color evolution, nutrient accumulation and other ripening-related processes of non-climacteric fruits. For instance, ethylene treatment accelerates grape fruit ripening by promoting coloration and ethylene production, increasing phytochemical contents and decreasing chlorophyll and titratable acid contents [41,42]. The exogenous ethylene-triggered coloration in the fruit peel increases with the ripening of Navelate orange, since ethylene stimulates carotenoid accumulation and reduces the chloroplastic carotenoid contents [43]. In litchi, ethylene plays a more important role in chlorophyll degradation in comparison with abscisic acid [44]. During blueberry ripening and storage, the role of ethylene in pigment and texture changes is genotype-dependent [45,46]. Nevertheless, the progress concerning the ethylene-induced molecular change in non-climacteric ripening is still slow, although extensive literature research has been reported on ethylene regulating non-climacteric ripening from physiological aspects. In addition, multiple research studies have spotlighted ethylene biosynthesis, while few studies have systematically focused on ethylene signal transduction.
Strawberry is a model non-climacteric fruit crop with a lack of ethylene burst during the ripening process. It has been documented that ethylene could affect some aspects of strawberry ripening. Therefore, the identification of ethylene signaling pathway genes in strawberry is necessary for fruit ripening study and breeding. In this study, a total of 29 ethylene signal transduction genes including 15 FaETRs and 14 FaEIN3/EINs were characterized from the octoploid strawberry genome, which was more than the number existing in the genomes of many fruit species: four ETRs and five EIN3/EINs in woodland strawberry [13,47], four ETRs and four EIN3/EINs in mulberry [48], four ETRs and four EIN3/EINs in peach [49], seven ETRs and nine EIN3/EINs in tomato [13,50], four ETRs and four EIN3/EINs in grape [51] and other fruit trees. This finding suggested that the FaETR and FaEIN3/EIN gene family has undergone an expansion in the octoploid strawberry. However, diverse expression patterns were observed between members of the FaETR and FaEIN3/EIN gene family, suggesting that there are different regulatory systems between the members of these gene families affecting their expression levels [52,53]. The ETRs from strawberry, Arabidopsis and tomato in the phylogenetic tree were divided into two subfamilies, which was consistent with previous reports [25,48]. The ETR classification in subfamily 1 or subfamily 2 relies on the non-functional HATPase_c domain present in the subfamily 2 members [25]. FaETRs and FaEIN3/EINs in the same cluster shared similar conserved motifs and exon–intron structures, while there existed differences in gene structure and motifs among different clusters. Multiple research projects have suggested that structural diversity is the primary resource for the evolution of multigene families [36,54,55]. Also, based on phylogeny analysis, it seems that the expansion and diversity among ETR and EIN3/EIN gene family members occur more after the divergence of monocot and dicot [56].
To investigate the potential function of FaETR and FaEIN3/EIN genes during strawberry fruit development and ripening, RNAseq datasets that were produced and validated in previous studies [37,38] were used to analyze the expression changes of these genes. The results showed that most FaETRs had a higher expression at the onset of fruit ripening in three strawberry cultivars, in line with the results of several studies on other non-climacteric fruits like grape [25,57] and citrus [25]. It has been reported that ETR genes seem to be more responsive in younger strawberries than in older ones during fruit ripening, since they showed a great expression increase between stages large green and white [35]. EIN3/EINs belong to a small family of transcription factors that activate the downstream components (ethylene-responsive factors and other downstream genes) of the ethylene signaling pathway [58,59,60]. Specific FaEIN3/EINs showed high expression levels at the initial red stage of the strawberry fruit in a certain cultivar, suggesting they might play a role during fruit ripening. Also, Ma et al. [61,62] proposed that LcEIL2 and LcEIL3 regulated ethylene-activated litchi fruitlet abscission by binding to LcCEL2/8 and LcPG1/2 in a cell wall remodeling process and LcACS1/4/7 and LcACO2/3 in the ethylene biosynthetic pathway. Mu et al. (2016) [57] demonstrated that the expression levels of most of the genes involved in ethylene biosynthesis and signaling had the same changing trend in non-climacteric fruits (grape and strawberry): they reached a maximum in the fruit development and then decreased, indicating that the mechanism of ethylene production and perception occurs in these fruits prior to ripening.
It has been well-documented that the ABA signaling pathway plays a central role in non-climacteric fruit ripening, including strawberry, grape and sweet cherry [63,64]. Multiple lines of evidence have shown that ethylene and ABA worked in synergy during non-climacteric fruit ripening; ethylene signaling could be involved in adjusting non-climacteric fruit ripening by merging into the ABA signaling pathway [20,21,44]. Jiang et al. (2003) [65] reported that exogenous ABA accelerated fruit coloring and softening through the up-regulation of ethylene production and PAL activity. Here, the expression levels of two FaETRs (FaETR2 and FaETR13) and two FaEINs (FaEIN2 and FaEIN7) were induced by ABA and inhibited by NDGA, indicating they might integrate ethylene and ABA signaling to regulate strawberry fruit ripening.

5. Conclusions

In summary, a total of 15 FaETRs and 14 FaEIN3/EINs in the ethylene signaling pathway from the cultivated strawberry were screened. Subsequently, a systematic analysis, including the chromosome location, evolutionary relationship, gene structure, conserved motif and protein–protein interaction network, was performed. Phylogenetic analysis showed that FaETRs and FaEIN3/EIL genes were respectively divided into two and three classes. The closely related proteins in the same class exhibited similar gene structure and motif composition. Furthermore, the expression profiles of FaETRs and FaEIN3/EIL genes during fruit ripening and in response to abscisic acid indicated that some of them in the ethylene signaling pathway probably participated in the strawberry ripening process. This work provides a landscape of strawberry ethylene signaling pathway gene families and a basis for further studies on strawberry breeding.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy13071930/s1, Table S1: Primers used for qRT-PCR.

Author Contributions

Conceptualization, H.T. and Y.Z. (Yunting Zhang); software, X.G. and C.G.; validation, M.D. and Y.C.; methodology, Y.Z. (Yunting Zhang) and C.G.; formal analysis, Y.L. (Yuanxiu Lin) and Y.W.; visualization, Y.Z. (Yunting Zhang) and C.G.; writing—original draft preparation, Y.Z. (Yunting Zhang), M.D. and X.G.; writing—review and editing, Y.Z. (Yong Zhang), Q.C., Y.L. (Ya Luo) and X.W.; funding acquisition, H.T. and Y.Z. (Yunting Zhang). All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Double Support Project of Discipline Construction of Sichuan Agricultural University (03573134) and the Natural Science Foundation of Sichuan Province (2023NSFSC1243).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Chromosome distribution of FaETR genes (A) and FaEIN3/EIN genes (B) in strawberry.
Figure 1. Chromosome distribution of FaETR genes (A) and FaEIN3/EIN genes (B) in strawberry.
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Figure 2. Phylogenetic analysis of FaETR family (A) and FaEIN3/EIN family (B) among different plant species. Red stars indicated the FaETRs or FaEIN3/EINs in strawberry. Fa, Fragaria × ananassa; At, Arabidopsis thaliana; Solyc, Solanum lycopersicum; Os, Oryza sativa; GSVIVT, Vitis vinifera.
Figure 2. Phylogenetic analysis of FaETR family (A) and FaEIN3/EIN family (B) among different plant species. Red stars indicated the FaETRs or FaEIN3/EINs in strawberry. Fa, Fragaria × ananassa; At, Arabidopsis thaliana; Solyc, Solanum lycopersicum; Os, Oryza sativa; GSVIVT, Vitis vinifera.
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Figure 3. Gene structure and conserved motif of FaETRs (A) and FaEIN3/EINs (B). Exons and introns are indicated by filled yellow boxes and single lines, respectively. Conserved motifs are indicated by different color boxes numbered 1–15.
Figure 3. Gene structure and conserved motif of FaETRs (A) and FaEIN3/EINs (B). Exons and introns are indicated by filled yellow boxes and single lines, respectively. Conserved motifs are indicated by different color boxes numbered 1–15.
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Figure 4. A protein–protein interaction network for FaETRs and FaEIN3/EINs in ethylene signaling pathway. XP_004303028.1, Serine/threonine-protein kinase CTR1-like (CTR1); XP_004287307.1, EIN3-binding F-box protein 1-like (EBF1); XP_004291457.1, EIN3-binding F-box protein 1-like (EBF1); XP_004291036.1, Protein REVERSION-TO-ETHYLENE SENSITIVITY1-like (RTE1); XP_004306246.1, Ethylene-insensitive protein 2-like (EIN2); XP_004287852.1, Protein RTE1-HOMOLOG-like (RTE1); XP_004294685.1, Mitogen-activated protein kinase 3-like (MAPK3); XP_004287157.1, Mitogen-activated protein kinase homolog MMK1-like (MAPK1); XP_004296361.1, Ethylene-responsive transcription factor 1B-like (ERF1B); XP_004302079.1, Ethylene-responsive transcription factor 1B-like (ERF1B). The red color text was indicated as the corresponding putative orthologous genes in the octoploid strawberry.
Figure 4. A protein–protein interaction network for FaETRs and FaEIN3/EINs in ethylene signaling pathway. XP_004303028.1, Serine/threonine-protein kinase CTR1-like (CTR1); XP_004287307.1, EIN3-binding F-box protein 1-like (EBF1); XP_004291457.1, EIN3-binding F-box protein 1-like (EBF1); XP_004291036.1, Protein REVERSION-TO-ETHYLENE SENSITIVITY1-like (RTE1); XP_004306246.1, Ethylene-insensitive protein 2-like (EIN2); XP_004287852.1, Protein RTE1-HOMOLOG-like (RTE1); XP_004294685.1, Mitogen-activated protein kinase 3-like (MAPK3); XP_004287157.1, Mitogen-activated protein kinase homolog MMK1-like (MAPK1); XP_004296361.1, Ethylene-responsive transcription factor 1B-like (ERF1B); XP_004302079.1, Ethylene-responsive transcription factor 1B-like (ERF1B). The red color text was indicated as the corresponding putative orthologous genes in the octoploid strawberry.
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Figure 5. Expression levels of FaETR and FaEIN3/EIN genes in strawberries. (A) Expression levels of FaETRs in three strawberry cultivars at different fruit developmental stages. (B) Expression levels of FaEINs in three strawberry cultivars at different fruit developmental stages. (C) Expression levels of FaETRs in strawberries after ABA and NDGA treatment. (D) Expression levels of FaEINs in strawberries after ABA and NDGA treatment. CK0, strawberries treated with distilled water on day 0; CK5, ABA5 and NDGA5, strawberries treated with distilled water, ABA and NDGA on day 5, respectively; CK8, strawberries treated with distilled water, ABA and NDGA on day 8, respectively.
Figure 5. Expression levels of FaETR and FaEIN3/EIN genes in strawberries. (A) Expression levels of FaETRs in three strawberry cultivars at different fruit developmental stages. (B) Expression levels of FaEINs in three strawberry cultivars at different fruit developmental stages. (C) Expression levels of FaETRs in strawberries after ABA and NDGA treatment. (D) Expression levels of FaEINs in strawberries after ABA and NDGA treatment. CK0, strawberries treated with distilled water on day 0; CK5, ABA5 and NDGA5, strawberries treated with distilled water, ABA and NDGA on day 5, respectively; CK8, strawberries treated with distilled water, ABA and NDGA on day 8, respectively.
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Figure 6. Expression levels of selected FaETRs and FaEIN3/EINs in ‘Xiaobai’ strawberry. (A) Expression levels of FaETR2 at different fruit developmental stages. (B) Expression levels of FaETR13 at different fruit developmental stages. (C) Expression levels of FaEIN2 at different fruit developmental stages. (D) Expression levels of FaEIN7 at different fruit developmental stages.BG, big green; TR, turn red; FR, full red. The data are presented as mean ± standard error (SE) analyzed using IBM SPSS Statistics 23.0 and one-way ANOVA. Duncan’s multiple-range test was employed to determine the differences in gene expression levels among different fruit developmental stages at the significance level of p ≤ 0.05.
Figure 6. Expression levels of selected FaETRs and FaEIN3/EINs in ‘Xiaobai’ strawberry. (A) Expression levels of FaETR2 at different fruit developmental stages. (B) Expression levels of FaETR13 at different fruit developmental stages. (C) Expression levels of FaEIN2 at different fruit developmental stages. (D) Expression levels of FaEIN7 at different fruit developmental stages.BG, big green; TR, turn red; FR, full red. The data are presented as mean ± standard error (SE) analyzed using IBM SPSS Statistics 23.0 and one-way ANOVA. Duncan’s multiple-range test was employed to determine the differences in gene expression levels among different fruit developmental stages at the significance level of p ≤ 0.05.
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Table
Table 1. Information of FaETR genes identified in strawberry.
Table 1. Information of FaETR genes identified in strawberry.
NameProtein
Size		MW (Da)Theoretical pIInstability IndexAliphatic IndexGRAVYSubcellular Localization
FaETR174183,123.006.9749.44108.490.120ER
FaETR274183,037.966.8249.72109.010.135ER
FaETR374183,162.037.1549.57108.100.106ER
FaETR463370,886.336.1136.77105.340.109ER
FaETR563370,856.215.8336.41105.340.104ER
FaETR663370,805.216.0436.21105.040.117ER
FaETR755362,261.505.9237.50108.050.126ER
FaETR874183,152.047.1449.01108.350.116ER
FaETR976584,496.326.6640.47103.200.132ER
FaETR1076584,601.376.4940.22102.300.123ER
FaETR1176584,544.396.4940.37102.300.124ER
FaETR1277486,848.736.3140.4197.070.017ER
FaETR1376986,328.166.8044.4397.200.013ER
FaETR1477487,072.147.2544.5596.450.002ER
FaETR1577486,920.846.4142.3897.070.016ER
Table
Table 2. Information of FaEIN3/EIN genes identified in strawberry.
Table 2. Information of FaEIN3/EIN genes identified in strawberry.
NameProtein
Size		MW (Da)Theoretical pIInstability IndexAliphatic IndexGRAVYSubcellular Location
FaEIN161669,685.515.4147.2661.09−0.690nucleus
FaEIN261870,050.055.4747.8962.14−0.677nucleus
FaEIN361869,906.895.5246.8661.50−0.675nucleus
FaEIN461769,732.665.5347.9162.40−0.668nucleus
FaEIN560267,753.365.0949.9565.76−0.633nucleus
FaEIN660267,664.215.1750.0364.80−0.657nucleus
FaEIN759366,308.725.6256.7671.21−0.732nucleus
FaEIN859366,332.725.5053.8370.56−0.732nucleus
FaEIN979788,258.076.1351.4777.06−0.533nucleus
FaEIN1059066,162.565.6855.0870.42−0.749nucleus
FaEIN1144950,979.555.1043.4077.71−0.607nucleus
FaEIN1223226,951.895.9455.4085.30−0.674nucleus
FaEIN1340846,862.975.1146.5373.55−0.682nucleus
FaEIN1427531,453.796.2043.5161.35−0.589nucleus
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Zhang, Y.; Deng, M.; Gu, X.; Guo, C.; Chen, Y.; Lin, Y.; Chen, Q.; Wang, Y.; Zhang, Y.; Luo, Y.; et al. Ethylene Signaling Pathway Genes in Strawberry and Their Expression Patterns during Fruit Ripening. Agronomy 2023, 13, 1930. https://doi.org/10.3390/agronomy13071930

AMA Style

Zhang Y, Deng M, Gu X, Guo C, Chen Y, Lin Y, Chen Q, Wang Y, Zhang Y, Luo Y, et al. Ethylene Signaling Pathway Genes in Strawberry and Their Expression Patterns during Fruit Ripening. Agronomy. 2023; 13(7):1930. https://doi.org/10.3390/agronomy13071930

Chicago/Turabian Style

Zhang, Yunting, Meiyi Deng, Xianjie Gu, Chenhui Guo, Yan Chen, Yuanxiu Lin, Qing Chen, Yan Wang, Yong Zhang, Ya Luo, and et al. 2023. "Ethylene Signaling Pathway Genes in Strawberry and Their Expression Patterns during Fruit Ripening" Agronomy 13, no. 7: 1930. https://doi.org/10.3390/agronomy13071930

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