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Article

Identification of AP2/ERF Transcription Factors and Characterization of AP2/ERF Genes Related to Low-Temperature Stress Response and Fruit Development in Luffa

by
Jianting Liu
1,†,
Haifeng Zhong
1,†,
Chengjuan Cao
2,†,
Yuqian Wang
1,
Qianrong Zhang
1,
Qingfang Wen
1,
Haisheng Zhu
1 and
Zuliang Li
1,*
1
Fujian Key Laboratory of Vegetable Genetics and Breeding, Crops Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China
2
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(11), 2509; https://doi.org/10.3390/agronomy14112509
Submission received: 10 September 2024 / Revised: 18 October 2024 / Accepted: 24 October 2024 / Published: 25 October 2024
(This article belongs to the Section Crop Breeding and Genetics)
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Abstract

Plant-specific APETALA2/Ethylene-Responsive Factor (AP2/ERF) transcription factors are involved in the regulation of genes associated with the growth and developmental processes of numerous plants. Although AP2/ERF proteins from other species have been intensively studied, no studies have been reported on the AP2/ERF family of Luffa cylindrica, an important vegetable of the cucurbit family, and one of the most popular vegetables in the world. In this study, 133 genes (315–6696 bp) encoding LcAP2/ERF proteins with complete AP2/ERF domains were identified according to the luffa P93075 genome. These LcAP2/ERF genes were subsequently classified and analyzed for their gene structures, chromosomal distribution locations, promoter cis-acting elements, conserved structural domains of encoded proteins, and responses to abiotic stresses. The LcAP2/ERF genes were identified and divided into five phylogenetic groups (AP2, DREBs, ERFs, RAV, and soloists). These genes were unevenly distributed across 13 chromosomes. An analysis of gene structures indicated the LcAP2/ERF genes contained 0–11 introns (average of 4.4). Additionally, 16 motifs were identified in the LcAP2/ERF proteins that were conserved across different phylogenetic groups. Moreover, 11 cis-acting elements associated with response to the environment were analyzed in a 2000 bp region upstream of the LcAP2/ERF gene promoters. A transcriptome analysis involving RNA-seq data revealed tissue-specific LcAP2/ERF expression profiles and the diversity in LcAP2/ERF expression. The effects of low-temperature stress on LcAP2/ERF expression were determined. Furthermore, fruit-development-related and low-temperature-induced expressional changes were verified by RT-qPCR analyses of 14 differentially expressed LcAP2/ERF genes in luffa. Our findings will help clarify the evolution of the luffa AP2/ERF family, while also providing valuable insights for future studies on AP2/ERF functions.

1. Introduction

In plants, the APETALA2/Ethylene-Responsive Factor (AP2/ERF) superfamily is one of the largest families of transcription factors (TFs), with each family member containing at least one highly conserved AP2/ERF DNA-binding domain comprising 50–60 amino acid residues [1,2,3,4,5]. According to the number of structural domains and the amino acid sequence similarity of AP2/ERF protein, AP2/ERF TFs are usually categorized into the following five subfamilies: DREBs (dehydration-responsive element-binding proteins), ERF (ethylene-responsive element-binding proteins), AP2 (APETALA2), RAV (related to ABI3/VP), and soloists (a few unclassified AP2/ERF TFs) [4,6,7,8,9,10]. These type of TFs are extensively involved in defense responses to a diverse range of biotic stresses, as well as the control of both growth [11] and development [12] in response to pathogen-induced stimuli [13,14], drought [15,16,17], salinity [18], heat [19], and cold [20]. It was demonstrated in early studies that AP2/ERF proteins are critical for decreasing stress-induced damages and enhancing stress tolerability via signal transduction pathways. Moreover, they are able to produce a wide variety of proteins that mediate plant defense responsiveness [5,21]. Numerous studies have been conducted on the functional characterization of AP2/ERF-encoding genes in both model plants and some important and commonly grown crops. For instance, in Arabidopsis thaliana [3], rice [22], wheat [19], soybean [16], tomato [23], cucumber [24], maize [12], and Chinese prickly ash [10], the function of AP2/ERF TFs has been linked to the plant’s response to abiotic stresses, such as low-temperature conditions. In addition, AP2/ERF TFs are also widely involved in regulating the development of various fruits, including pomegranate [25], Chinese jujube [26], sweet cherry (Prunus avium) [11], and strawberry [27].
Low temperature is considered a mostly abiotic stress factor affecting plant growth, the development process, and physiological and ecological traits [1,28]. Studies have shown that continuous low-temperature conditions for cucurbit crops before flowering will cause hazards such as flower topping, poor fertilization, reduced fruit set, etc. Low temperature encountered during the seedling stage often slows down root growth and shortens plants, which seriously affects the distribution of crops, their yields, and their qualities [29,30,31]. During cultivation, luffa [Luffa cylindrica Roem. (smooth)] plants are sensitive to extreme weather conditions, such as low temperatures and sparse sunlight. Such conditions cause flowers and fruits to fall, while also leading to seedling death and various diseases, with detrimental effects on luffa production [32,33]. In the process of luffa production, low-temperature disasters lead to luffa yield reductions of 20% to 50%, and in severe cases, this can be up to 70%, and even cause the extinction of the harvest. According to previous research, many AP2/ERF TFs participate in the regulation of plant growth and development in different environments [10,34,35,36,37]. The overexpression of HcTOE3, which belongs to the AP2 subfamily in Halostachys caspica, enhances the tolerance of A. thaliana to cold stress by upregulating the transcription of CBF1 and COR15 [38]. In Vitis amurensis, the ethylene response factor VaERF092 regulates the expression of VaWRKY33 by directly binding to its promoter region GCC-box, and thus enhances low-temperature resistance [39]. In Poncirus trifoliata, PtrERF108 can increase cold tolerance by modulating raffinose synthesis via its regulatory effects on PtrRafS expression [40]. Moreover, luffa fruit size and shape are important agronomic traits that affect the yield and quality of harvested organs. However, the mechanism regulating these traits is unknown in luffa and other Cucurbitaceae crops. Interestingly, AP2/ERF TFs help regulate fruit development (e.g., size, length, and ripening) [10,11,23,26,27]. In Solanum lycopersicum, ENO, which is an AP2/ERF TF, binds to the SlWUS promoter region to directly regulate expression and control tomato fruit size [41]. However, in sweet cherry, a type of AP2/ERF TF (PavRAV2) negatively influences fruit size by directly suppressing the expression of PavKLUH [11]. However, research reports related to the important commercial traits of fruit size and shape have not yet been reported in luffa. Meanwhile, due to the lack of effective genetic resources, research into low-temperature stress response and fruit-development-related molecular biology is also a blank slate for luffa.
The genomes of many plant species have been sequenced, including A. thaliana [42], Oryza sativa [43], Zea mays [44], Lycopersicon esculentum [45], Saccharum spontaneum [46], Solanum melongena [47], Brassica rapa [48], Ziziphus jujuba [49], Gossypium sp. [50], and Malus domestica [51]. Genome sequences of Cucurbitaceae crops, such as Benincasa hispida [52], Citrullus lanatus [53], Cucurbita máxima [54], Cucumis melo [55], Cucurbita pepo [56], and L. cylindrica Roem. [57], have recently been released. The publication of the genome of luffa is of significant importance for the study of cucurbit species for genome-wide analysis. In this study, gene expression patterns were comprehensively analyzed using bioinformatics techniques and methods. Several LcAP2/ERF gene-encoded proteins were identified as candidate transcription factors involved in abiotic stress response and fruit development in luffa plants based on their tissue expression profiles, as well as their response to low- temperature stress and their expression patterns related to fruit development.

2. Materials and Methods

2.1. Plant Materials, Growth Conditions, and Stress Treatment

We dipped rounded-end clipped shells of Fusi-1 luffa seeds in warm water at 50 °C for half an hour, then rinsed them three times with double-distilled water, sterilized them in 75% alcohol solution for 10 min, rinsed them three times with distilled water, and then sowed them in 32-hole cavity trays. The seedling trays of sown luffa seeds were then placed in a light incubator for seedling culture with the following conditions: 12 h day (28 °C)/12 h night (25 °C); light intensity controlled to 300 μmol photons m−2 s−1; and a relative humidity of 75%. About two-week-old luffa seedlings (two-leaf, one-center stage) were low-temperature-treated at 5 °C for 0, 2, 4, 8, and 12 h, controlling the light intensity to 80 μmol photons m−2 s−1 as previously described. The 2nd true leaf was collected as material for subsequent experimental study, and the 0 h samples without low-temperature treatment were used as controls [33]. Leaf chlorophyll fluorescence images were obtained using the IMAGING-PAM-MAX chlorophyll fluorescence imaging platform (Walz, Effeltrich, Germany), and luffa seedlings were dark-adapted before irradiation with saturated pulsed light (12,000 μmol photons m−2 s−1) for 30 min to detect variable fluorescence (Fv) and maximum fluorescence (Fm) in the dark-acclimated leaves and to calculate the maximum photochemical quantum efficiency (Fv/Fm) values (Supplementary File S1). Moreover, the luffa hybrid Fuyan-3 (local main cultivar) was selected as the experimental material for analyzing fruit development (Supplementary File S2). Uniformly developing fruits were collected 2 days before flowering (i.e., ovary) and 0, 2, 4, 6, 8, and 10 days after flowering from the Dongzhang Base of the Crop Research Institute of Fujian Academy of Agricultural Sciences (north of 25.71° N and east of 119.25° W) in June 2022. All samples, both control and treated, were examined with three bio-replicates. Then, the luffa leaf samples and fruit samples were frozen in liquid nitrogen and stored at −80 °C immediately after being combined.

2.2. Total RNA Extraction, RNA Sequencing, and Gene Expression Analysis

The study used the Trizol method to extract the total RNA from the leaves of the luffa seedlings, and each sample was taken in equal quantities and mixed to form three RNA pools. These were analyzed and the concentration, purity, and completeness of the total RNA samples of luffa in sequence were detected using Qubit 2.0, Nanodrop, and Aglient 2100. The cDNA library construction of luffa was entrusted to Suzhou BioNovoGene Technologies Co. (Suzhou, China), as previously described [58], and then the transcriptome sequencing analyses were performed using the Illumina HiSeq 2500 platform. High-quality reads obtained after the completion of sequencing were aligned with the reference genome of luffa (PRJNA596077), using Bowtie2 software (version 2.5.3) to obtain transcript or gene annotations [59].
Tissue-specific LcAP2/ERF expression profiles and the effects of low-temperature stress were investigated using our publicly available Fusi-1 (PRJNA1044273) transcriptome data [33]. To obtain the results of low-temperature stress in luffa leaves and in different periods of fruit development, the FPKM values were calculated using StringTie2 software (v.2.1.5) [60]. For the low-temperature treatment, control plants were incubated at 25 °C, whereas the treated plants were incubated at 5 °C, with samples collected at 2, 4, 8, and 12 h. The transcript level of each gene was calculated relative to its corresponding transcript level in the control plants. For the fruit development period, Fuyan-3 samples collected at 2 days before flowering and 0, 2, 4, 6, 8, and 10 days after flowering were examined.
For the RT-qPCR assay, 14 pairs of quantitative primers (i.e., LcAP2/ERF-Fq and LcAP2/ERF-Rq) were designed based on the coding sequence (i.e., CDS) of luffa in the present study (Supplementary File S3). The RT-qPCR program used to amplify the internal control gene (Lc18S rRNA) and the method used to calculate relative gene transcript levels have been described previously [58,61].

2.3. The Identification of LcAP2/ERF Genes from Genome of the Luffa

LcAP2/ERF gene family members, identified on the basis of the L. cylindrica cultivar P93075 reference genome, were downloaded from the NCBI database (accessed on 30 June 2024) [57]. Information related to the luffa genome assembly was downloaded to construct a local database. This information included LcAP2/ERF genes, their promoter and coding sequences, and the amino acid sequences of the encoded proteins.

2.4. Bioinformatics Analysis of the LcAP2/ERF Gene Family

All potential LcAP2/ERF genes were confirmed by HMMER analysis. The LcAP2/ERF gene structures, promoter sequences, and Ka/Ks ratios for the duplicated genes [62], as well as the phylogenetic relationships, conserved motifs, pI, molecular weights, and subcellular localization of the putative LcAP2/ERF proteins were analyzed as previously described [33,61].

2.5. Subcellular Localization of LcAP2/ERF Transcription Factors

To examine the subcellular localization of two LcAP2/ERF (i.e., LcAP2/ERF-71 and LcAP2/ERF-107) proteins, the appropriate full-length LcAP2/ERF sequences without stop codons were amplified by polymerase chain reaction (i.e., PCR) using the primers (with Bam HI cleavage sites) presented in Supplementary File S3. The PCR-amplified and correctly sequenced LcAP2/ERF-71 and LcAP2/ERF-107 gene sequences were ligated by homologous recombination into the transient expression vector pCAMBIA1300, an empty vector with a GFP tag. The constructed recombinant plasmids pCAMBIA1300-LcAP2/ERF-71 (35S::LcAP2/ERF-71::GFP) and pCAMBIA1300-LcAP2/ERF-107 (35S::LcAP2/ERF-107::GFP) were introduced into the leaves of N. benthamiana seedlings via A. tumefaciens (GV3101)-mediated transformation for an analysis of subcellular localization. The empty vector pCAMBIA1300-GFP (35S::GFP) was similarly introduced as the control. The leaf cells on the underside of the tobacco leaves were examined with a laser confocal scanning microscope to detect GFP fluorescence, as previously described [33].

3. Results

3.1. Characterization of LcAP2/ERF GENES in Luffa

In this study, 133 AP2/ERF proteins with complete structural domains were identified from the whole-genome data of luffa. Based on the order of these AP2/ERF protein-coding genes on the 13 luffa chromosomes, the LcAP2/ERF genes were named LcAP2/ERF-1–133 (Supplementary File S4) [57]. The coding sequence lengths ranged from 234 to 2250 bp (Supplementary File S5). The molecular weights of the encoded proteins ranged from 11.15 kDa (LcERF-19) to 84.96 kDa (LcERF-60), with an average of 30.50 kDa (Supplementary File S6). Only 11 LcAP2/ERF proteins had a molecular weight exceeding 50 kDa (LcAP2/ERF-34, LcAP2/ERF-70, LcERF-73, LcAP2/ERF-110, LcAP2/ERF-2, LcAP2/ERF-74, LcAP2/ERF-125, LcAP2/ERF-44, LcAP2/ERF-80, LcAP2/ERF-11, and LcERF-60). The isoelectric point (pI) ranged from 4.11 (LcERF-64) to 10.47 (LcERF-98), with an average of 6.93. Of the identified LcAP2/ERF proteins, 41.35% (55/133) had a pI greater than 7.0. The results of the subcellular localization analysis suggested that 105 LcERF TFs were located in the nucleus (78.9%), 17 LcERF TFs were located in the chloroplast (12.7%), 5 LcERF TFs were located in the cytoplasm (3.7%), and 4 LcERF TFs were located in the mitochondria (3.0%). Notably, LcERF-100 and LcERF-128 were revealed as extra-mitochondrial proteins (Supplementary File S4).

3.2. The Chromosome Localization and Replication of LcAP2/ERF Genes

Using MapChart software (version 2.3.2) analyses, we mapped the above 133 LcAP2/ERF genes to 13 luffa chromosomes (Figure 1). Chromosome 2 contained the most LcAP2/ERF genes (15), followed by chromosomes 1 and 5 (13 each), whereas only 21 LcAP2/ERF genes were located on chromosomes 6, 8, and 13 (7 each). The other chromosomes contained 8–12 LcAP2/ERF genes. Genome and genetic system evolution is known to be motivated by gene duplication events (segmental and tandem duplications) [63]. A tandem duplication involves a chromosomal region comprising two or more genes within a 200 kb range [64]. Gene duplication events have contributed greatly to the expansion and evolution of LcAP2/ERF genes, especially via segmental duplications [2]. To investigate the mechanism by which the LcAP2/ERF gene undergoes differentiation after replication and to research the selective effects, we then computed the non-synonymous replacement rate (Ka) and synonymous replacement rate (Ks) as well as the nucleotide replacement rate (Ka/Ks ratio) for tandem and segmental duplications (Supplementary File S7). In this study, the Ka/Ks ratio for tandemly (LcERF-128/LcERF-129) and segmentally (LcERF-21/LcERF-65 and LcERF-29/LcERF-62) duplicated genes ranged from 1.1407 to 50, suggesting these six genes belonging to the AP2/ERF family evolved under positive selection pressure [65].

3.3. Promoter Cis-Acting Elements and Structural Analyses of LcAP2/ERF Genes

To determine the distribution of cis-acting elements present in the promoters of the LcAP2/ERF genes, the sequence of each LcAP2/ERF gene promoter (2.0 kb upstream region) was analyzed using the PlantCARE database (Supplementary File S8). A total of 20,568 (92.79%) known cis-acting elements were identified in the 133 luffa LcAP2/ERF promoters. The analysis revealed that these cis-acting elements are relevant to both abiotic and biotic stress responses, as well as physiological and developmental progression. Specifically, a total of 1571 light-responsive elements and 356 anaerobic-induced elements were detected in 100.00% and 91.73% of the LcAP2/ERF promoter regions, respectively (Figure 2a); these were the most abundant cis-acting elements among the 11 identified elements (Supplementary File S9). Additionally, there were 291 ABA-responsive elements and 404 MeJA-responsive elements identified in 75.19% and 74.44% of the LcAP2/ERF gene promoter regions, respectively. In total, 82 MYB-binding sites, 95 gibberellin-responsive elements, and 78 SA-responsive elements were detected in 46.62%, 45.11%, and 42.11% of the LcAP2/ERF promoter regions, respectively. Moreover, 72 auxin-responsive elements were detected in 39.10% of the LcAP2/ERF promoter regions, while 62 defense and stress-responsive elements were identified in 40.60% of the LcAP2/ERF promoter gene areas, and 74 low-temperature-responsive elements were detected in 38.35% of the LcAP2/ERF promoter regions. In addition, 14 wound-responsive elements were also identified in 14 LcAP2/ERF promoter regions. The presence of these multifunctional cis-acting elements reflects the functionally diverse TFs encoded by the LcAP2/ERF gene in luffa (Supplementary File S9).
The structural features of the LcAP2/ERF genes identified from luffa were analyzed using GSDS online software (version 2.0). The examination of the exon and intron regions (Figure 2b) revealed 63% of the 133 identified LcAP2/ERF genes had one exon. In addition, 23 LcAP2/ERF genes comprised 2 exons, 8 LcAP2/ERF genes contained 9 exons, 4 LcAP2/ERF genes included 7 exons, 4 LcAP2/ERF genes had 10 exons, 3 LcAP2/ERF genes contained 8 exons, 3 LcAP2/ERF genes contained 3 exons, and 2 LcAP2/ERF genes contained 4 exons. Notably, LcERF-69 and LcERF-130 contained 14 and 6 exons, respectively. The identified LcAP2/ERF genes also differed in size, ranging from 315 bp (LcERF-5 in the DREB subfamily) to 6696 bp (LcAP2/ERF-90 in the AP2 subfamily) (Supplementary File S4). The LcAP2/ERF gene sizes in the ERF subfamily ranged from 321 bp (LcERF-64) to 2194 bp (LcERF-45). The LcAP2/ERF gene sizes in the AP2 subfamily ranged from 2097 bp (LcERF-13) to 6696 bp (LcAp2/ERF-90). The LcAP2/ERF gene sizes in the DREB subfamily ranged from 315 bp (LcERF-5) to 5175 bp (LcERF-36). The LcAP2/ERF gene sizes in the RAV subfamily ranged from 327 bp (LcERF-60) to 1030 bp (LcERF-107). The soloist LcAP2/ERF gene sizes ranged from 441 bp (LcERF-24) to 5036 bp (LcERF-57).

3.4. The Protein Phylogenetic Relationships and Conservation Patterns of LcAP2/ERF Transcription Factors

Based on the phylogenetic analyses, we found that 133 LcAP2/ERF genes encoding AP2/ERF proteins were classified into five groups (AP2, DREBs, ERFs, RAV, and soloists) [10]. The AP2 subfamily consisted of 18 (13.5%) LcAP2/ERF genes. The ERF subfamily contained 69 (51.8%) LcAP2/ERF genes, which was more than the 39 (29.3%) LcAP2/ERF genes in the DREB subfamily. The RAV and soloist subfamilies comprised four and three LcAP2/ERF genes, respectively (Figure 3a and Supplementary File S4). To further study the diversities among the conservative structural domain (i.e., motifs) in the LcAP2/ERF protein sequences, the 16 conserved motifs in the 133 LcAP2/ERF proteins were predetermined with the Multiple Em for Motif Elicitation (MEME) software (version 5.5.7). Among the 16 predicted AP2/ERF motifs, 4 (i.e., motifs 1, 2, 3, and 4) were confirmed to correspond to the broadly distributed AP2/ERF domain, whereas the remaining 12 motifs, which were limited to certain phylogenetic groups, were considered to play an important role in nuclear localization or transcriptional regulation (Figure 3b and Supplementary File S10). Interestingly, beyond the AP2/ERF domain, similar motifs were shared by proteins in the same group. For example, motifs 5 and 6 were detected in five AP2 subfamily members (but not in LcERF-57). Motif 16 was exclusive to the DREB subfamily (i.e., LcERF-89, LcERF-128, and LcERF-129). Similarly, motif 9 was detected only on proteins belonging to the DREB subfamily (i.e., LcERF-17, LcERF-32, and LcERF-123,). Motif 10 was present only on ERF subfamily members (e.g., LcERF-8, LcERF-40, and LcERF-67). These findings were in accordance with the results of earlier studies on the Z. mays and Juglans mandshurica AP2/ERF families [4,20].

3.5. LcAP2/ERF Expression Profiles in Seven Tissues

Using publicly available transcriptome data, seven tissues of luffa (roots, stems, leaves, male flowers, female flowers, fruits, and ovaries) were critically analyzed, resulting in a comprehensive examination of the expression levels of all 133 LcAP2/ERF genes. The fragments per kilobase per million transcripts (i.e., FPKM) values identified from the sequencing data (RNA-seq) of the transcriptome revealed differences in 133 LcAP2/ERF gene expressions between the selected tissues. More concretely, the highest expression levels of LcAP2/ERF were found in male flowers, followed by female flowers, roots, stems, fruits, leaves, and ovaries, with FPKM values of 5269.22, 4550.91, 2074.57, 1304.53, 1178.94, 1094.93, and 911.03, respectively (Figure 4a). Among the 133 LcAP2/ERF genes, LcERF-5, LcERF-12, LcERF-19, LcERF-24, LcERF-66, LcERF-82, and LcERF-115 had extremely low or no expression (average FPKM value was 0) of the seven organizations inspected. The FPKM values for 51 LcAP2/ERF genes (e.g., LcERF-40, LcERF-41, and LcAP2/ERF-44) were all below 10, whereas the FPKM values for 46 LcAP2/ERF genes (e.g., LcERF-105, LcERF-122, and LcERF-53) ranged from 10.51 to 98.66 in the selected tissues, the mean value of which was 39.64. In addition, the FPKM values of the remaining 29 LcAP2/ERF genes were detected from seven luffa organizations. Moreover, seven LcAP2/ERF genes were expressed at the highest level in male flowers, particularly LcERF-25 (average FPKM value was 591.67), as well as LcERF-6 (average FPKM value was 520.19). Another five LcAP2/ERF genes (LcERF-78, LcERF-126, LcERF-108, LcAP2/ERF-70, and LcERF-123) were predominantly expressed in the female flower (FPKM values of 381.01, 323.13, 271.70, 236.64, and 223.62, respectively). The LcERF-132 (FPKM value of 128.95), LcERF-73 (FPKM value of 39.47), and LcERF-37 (FPKM value of 17.43) genes were preferentially expressed in the leaf. The LcERF-1 (FPKM value of 188.04) and LcERF-125 (average FPKM value was 77.13) genes were preferentially expressed in the luffa fruit (Supplementary File S11).

3.6. Expression Analyses of LcAP2/ERF During the Low-Temperature-Induced Stress Condition

AP2/ERF TFs are the one of the largest transactivator family members in plants, but they have also been shown to be extensively involved in the process of abiotic stress signaling pathways [35,66]. In the present study, RNA-seq analyses were employed to identify LcAP2/ERF expression profiles at five time points (i.e., 0, 2, 4, 8, 12 h) during low-temperature (5 °C) stress conditions. The heatmap (Figure 4b) showed that FPKM values demonstrated a gradual increase in the expression levels of LcAP2/ERF genes, with FPKM values of 789.03, 1783.21, 2088.96, 2390.62, and 2711.62 at 0, 2, 4, 8, and 12 h, respectively (Supplementary File S12). Among the 133 LcAP2/ERF genes, 14 (e.g., LcERF-5, LcERF-17, and LcERF-22) had undetectable expression levels (FPKM value of 0) at 0, 2, 4, 8, and 12 h. The FPKM values for 53 LcAP2/ERF genes (e.g., LcERF-7, LcERF-13, and LcAP2/ERF-74) were less than 10, whereas the FPKM values for 46 LcAP2/ERF genes (e.g., LcERF-3, LcERF-4, LcERF-9, LcERF-10, and LcERF-12) ranged from 10.45 to 96.53 at the five time points, with an average of 31.16. Under the low-temperature stress conditions, FPKM values were also determined for the other 34 LcAP2/ERF genes (e.g., LcERF-42, LcERF-57, LcERF-123, LcERF-76, LcERF-108, LcERF-105, LcERF-61, LcERF-15, LcERF-60, and LcERF-122), which FPKM values ranging from 133.73 to 3440.78.
To verify the RNA-seq data, 14 LcAP2/ERF genes, which are potentially associated with low-temperature tolerance, were selected for RT-qPCR analyses in this study (Figure 5). The analyses indicated significant differences in the expression levels of these 14 LcAP2/ERF genes, and a number of them (71.43%) were found to be highly expressed during low-temperature treatment. Specifically, the expression levels of LcERF-20, LcERF-45, LcERF-127, LcERF-128, and LcERF-132 peaked at 2 h, whereas LcERF-23, LcERF-71, and LcERF-107 were most highly expressed at 4 h. Notably, LcERF-113 and LcERF-114 were highly expressed only at 8 h, while the expression levels of LcERF-6, LcERF-48, LcERF-64, and LcERF-78 were virtually undetectable within 12 h of low-temperature treatment. There was a high positive correspondence (R2 = 0.8791) with RT-qPCR and RNA-seq data, even with the LcAP2/ERF (e.g., LcERF-5, LcERF-17, LcERF-19, LcERF-22, LcERF-24, LcERF-27, LcERF-38) genes, which had a generally low level of expression (Figure 6).

3.7. Analysis of LcAP2/ERF Expression During Fruit Development in L. cylindrica

A RT-qPCR analysis was completed to examine the expression profiles of 14 LcAP2/ERF genes at seven different time points (i.e., at 2 days before flowering and 0, 2, 4, 6, 8, and 10 days after flowering) (Figure 7). There were significant differences in the expression levels of these 14 LcAP2/ERF genes, with numerous genes expressed highly among the luffa fruit development period. Specifically, the LcERF-6, LcERF-21, LcERF-64, and LcERF-78 expression levels peaked on day 2, whereas LcERF-107 and LcERF-132 were most highly expressed on day 4. Notably, LcERF-20, LcERF-71, and LcERF-114 were highly expressed only on day 8, whereas LcERF-45 and LcERF-48 were expressed at high levels only on day 10. In contrast, LcERF-127 and LcERF-128 expression levels were almost undetectable throughout the luffa fruit development period.

3.8. The Subcellular Localization of LcAP2/ERF Transcription Factors

To examine the subcellular localization of LcAP2/ERF transcription factors, we selected two LcAP2/ERF genes, LcAP2/ERF-71 and LcAP2/ERF-107, from the ERF and RAV subfamilies, respectively, and constructed transiently expressed fusion expression vectors in which they carried a GFP tag. The recombinant vectors were injected into tobacco (Nicotiana benthamiana) lower epidermal cells using Agrobacterium tumefaciens (GV3101)-mediated subcellular localization. The results showed that, in control cells containing the empty vector (35S::GFP), green fluorescent signals were detected throughout the cells harboring the 35S::LcAP2/ERF-71::GFP and 35S::LcAP2/ERF-107::GFP constructs; green fluorescence was detected exclusively in the nucleus. Accordingly, LcAP2/ERF transcription factors were localized to the nucleus, consistent with their predicted subcellular localization (Supplementary File S4 and Figure 8). These results imply that LcAP2/ERF-71 and LcAP2/ERF-107 proteins function as transcription factors in the nucleus.

4. Discussion

4.1. Characterization of Luffa LcAP2/ERF Genes

The AP2/ERF TF family is extensively involved in plant growth, development, signal transduction, and biotic and abiotic stress response functions [5,12,17,67]. Consequently, in the present study, we performed a genome-wide analysis and screened for 133 LcAP2/ERF genes (LcAP2/ERF-1–133) in L. cylindrica. Previous research indicated that the number of AP2/ERF transcription factors is not proportional to the genome size of a species [12,65,68,69]. More specifically, A. thaliana, rice, yellow horn, rose, and maize genomes contain 147, 165, 145, 135, and 184 AP2/ERF genes, but their genomes are 125, 430, 504, 600, and 2300 Mb in size, respectively. The number of AP2/ERF genes (i.e., 133) identified in the L. cylindrica genome (656 Mb) in the current study is consistent with the lack of a direct relationship between the number of AP2/ERF genes and genome size. Previous studies have also shown that cucurbits originated about 80 million years ago and that at least four whole-genome doubling (WGD) events have occurred during the genetic development of cucurbit crops. These WGD events may have been the primary factors contributing to the diversity of cucurbit phenology [70,71]. In the present study, a few gene duplication events involving LcAP2/ERF genes were detected in the L. cylindrica genome, which may explain the considerable abundance of AP2/ERF TFs in L. cylindrica (Supplementary File S2).
Phylogenetic analyses and the consequent clustering provided support for the categorization of the LcAP2/ERF genes into five groups (AP2, DREBs, ERFs, RAV, and soloists) based on the presence of AP2/ERF structural domains (Figure 3a). AP2/ERF genes were similarly classified in other plant species, including A. thaliana [68], poplar [72], and cucumber [24,34]. In grape, the AP2, DREB, ERF, RAV, and soloist groups consist of 18, 36, 73, 4, and 1 AP2/ERF TF genes [73]. There are currently 165 AP2/ERF proteins identified in rice, consisting of 24 members of the AP2 subfamily, 136 members of the ERF (ERF/DREB) subfamily, and 5 members of the RAV subfamily (i.e., no soloist subfamily members) [22]. The conserved AP2/ERF domains of LcAP2/ERF proteins were analyzed in this study. According to a previous study, AP2 group members have two duplicated AP2/ERF domains; this group can be further subdivided into AP2 and ANT subfamilies, which are involved in the development of female reproductive organs [74]. Proteins of the ERF and DREB subfamilies have only one AP2/ERF conserved structural domain; accordingly, based on the sequence of the DNA-binding structural domain, these proteins can be subdivided into two subfamilies, ERF and CBF/DREB, with ERF subfamily members binding to AGCCGCC core motifs and CBF/DREB subfamily members recognizing the cis-acting element A/GCCGAC (i.e., C-repeat) to modulate the plant defense response to abiotic stresses, including drought [17], low temperatures [37,75], and high salinity [67]. In addition, RAV proteins possess a VP1/ABI3 binding domain that enables them to bind specifically to target gene promoters with the consensus motif C(A/C/G)ACA(N)2-8(C/A/T)ACCTG [76]. And these AP2/ERF proteins can also play key regulatory roles in plant growth and development, as well as in defense pathways [34]. Therefore, it is worthwhile to further investigate the binding specificity and functionality of these LcAP2/ERF TFs that affect luffa growth and development, as well as abiotic stress response.

4.2. Analysis of L. cylindrica AP2/ERF Gene Promoters and Duplication Events

Studies have shown that phytohormone signaling pathways such as ABA, SA, and JA have important roles in the regulation of various biometabolic pathways in plants via transcription factors [77,78,79]. In the process of plant response to adversity stress, AP2/ERF transcription factors assume the functions of integrating upstream signals, amplifying the signals, and activating a series of stress-tolerant genes through transcriptional regulation, and are therefore important transcriptional regulators for low-temperature tolerance in plants [12,15,34,35,80,81,82]. In this study, various types of cis-acting elements (e.g., responsive to ABA, SA, auxin, and MeJA) were identified in LcAP2/ERF promoter regions, suggesting the TFs encoded by the LcAP2/ERF gene appears to be related to a variety of biological processes that regulate the growth and development of plants [12,17]. Previous studies have shown that, in tobacco, the overexpression of the JeRF1 gene results in a protein that can interact with multiple cis-acting elements and activate the expression of stress-responsive and ABA biosynthesis-related genes, ultimately leading to ABA biosynthesis, thereby enhancing tobacco stress tolerance and growth under low-temperature conditions [78]. In many cases, AP2/ERF expression is regulated by one or multiple conserved cis-acting elements present in the promoter region [3]. Earlier studies isolated plant-specific DNA-binding (GCC box) ERF TFs from tomato fruit (LeERF1–4) [83,84]. The GCC-box motif is sufficient for the SA-mediated suppression of JA-responsive gene expression [85]. In addition to defense responses, SA plays an important role in the response to abiotic stresses, including drought and low temperatures; the effects of exogenous SA depend on the concentration of the applied SA, the mode of application, and the state of the treated plants (e.g., developmental stage and acclimation) [36]. In A. thaliana, members of the ERF TF family typically bind to GCC-box motifs in the promoters of JA- and ethylene-responsive genes, thereby positively or negatively regulating their expression [86]. In apple, an AP2/ERF TF (MdABI4) integrates JA and SA signals to precisely modulate cold tolerance through the JAZ–ABI4–ICE1–CBF regulatory cascade [77]. A recent study showed that the auxin-induced degradation of the AP2/ERF TF ERF13, which depends on MPK14-mediated phosphorylation, is critical for lateral root development in A. thaliana [87]. During tomato fruit ripening, SlERF.D7 expression increases in response to exogenous ethylene and auxin treatments, most likely in a ripening inhibitor-independent manner [88]. In the present study, we identified various cis-acting elements in the LcAp2/ERF promoter in response to a large number of phytohormones (i.e., MeJA, ABA, auxin, and SA), which implies that LcAP2/ERF TFs may contribute to the control of a variety of hormone signaling pathways related to the developmental processes of luffa, as well as abiotic and biotic stress responses (Figure 2a and Supplementary File S9).
Gene duplication events may lead to the significant expansion of plant gene families. Tandem and segmental duplications are critical for plant development and responses to environmental stimuli. The findings of the current study suggest that only three pairs of replicated LcAP2/ERF genes (i.e., LcERF-128/LcERF-129, LcERF-21/LcERF-65, and LcERF-29/LcERF-62) were the result of tandem duplication events, and may have been the primary contributors to the expansion of the LcAP2/ERF gene family (Supplementary File S7). The subsequent analysis of Ka, Ks, and Ka/Ks values indicated that one tandemly and two segmentally duplicated AP2/ERF gene pairs in luffa had a Ka/Ks value exceeding 1. Intriguingly, the duplicated gene pairs LcERF-128/LcERF-129, LcERF-21/LcERF-65, and LcERF-29/LcERF-62 were subjected to a strong positive selection pressure during the evolution of the LcAP2/ERF family, implying that harmful mutations were actively eliminated, resulting in relatively diverse gene functions [89].

4.3. Expression Profiles of LcAP2/ERF Genes in L. cylindrica and the Changes in Expression Induced by Low-Temperature Stress

In this study, we explored LcAP2/ERF genes’ tissue expression patterns using transcriptomic data from seven tissues of L. cylindrica (root, stem, leaf, male flower, female flower, fruit, and ovary) (Figure 4a). Many of the selected LcAP2/ERF genes were highly expressed in the male flower (26.3%). Additionally, some LcAP2/ERF genes were highly expressed in the leaf (20.3%), root (19.5%), female flower (11.2%), stem (9.0%), ovary (5.2%), and fruit (3.0%). However, the expression of 5.2% of the LcAP2/ERF genes was undetectable in the seven examined tissues. These finding are similar to those of previous experiments studying other plants, including Punica granatum [25], Z. mays [4], and Rhododendron species [2]. Moreover, gene expression data revealed the tissue-specific expression profiles of the LcAP2/ERF genes, with potentially important roles in male luffa flower development. The expression levels of a few LcAP2/ERF genes (e.g., LcERF-6, LcERF-23, LcERF-29, LcERF-48, LcERF-77, LcERF-78, LcERF-107, and LcERF-129) peaked in flowers (i.e., male and female flowers) and the ovary, suggesting the critical function of the mechanism mediating the course of L. cylindrica flower formation and fruit development (Supplementary File S11. Furthermore, a few LcAP2/ERF genes were expressed at extremely low levels or not expressed in the seven tissues (e.g., LcERF-41, LcERF-42, LcERF-55, LcERF-59, LcERF-84, LcERF-87, and LcERF-95). These findings revealed that LcAP2/ERF transcription factors are differentially expressed at different levels in diverse tissues or organizations to mediate the diversified physiological and biological processes in luffa.
As a matter of fact, low-temperature stress can significantly affect the growth and development of plants. However, in the course of long-term genetic evolution, plants have developed a whole set of antioxidant protection systems controlled by superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione S-transferase (GST), glutathione peroxidase (GPX), and other reducing substances [90,91,92]. Transcription factors (ERFs) play an important role in the response to low-temperature stress, but the molecular mechanisms of this process are still poorly understood, and studies related to these molecular mechanisms are still only focused on model plants [10,75]. In Poncirus trifoliata, the overexpression of PtrERF9 significantly enhanced the low-temperature tolerance of transgenic strains by transcriptionally regulating PtrGSTU17 expression, as well as the key gene for ethylene synthesis, PtrACS, to increase GST enzyme activity, while decreasing the accumulation of ROS to achieve its role in the defense response to low-temperature stress in Poncirus trifoliata [81]. In the present study, we investigated the regulation of LcAP2/ERF TFs in low-temperature-treated luffa Fusi-1 seedlings (Supplementary File S1). An analysis of expression levels (i.e., FPKM values) under normal conditions (Figure 4a) showed that LcAP2/ERF genes (except LcAP2/ERF-132) were expressed at lower levels in the leaves than in the other examined tissues, with the exception of the ovary. Nevertheless, the increased expression level of LcAP2/ERF by 3.18-fold over the period from 0 h to 12 h under low-temperature conditions demonstrated the responsiveness of LcAP2/ERF genes to cold conditions. Furthermore, 14 LcAP2/ERF genes were analyzed by RT-qPCR experiments. The expression patterns of the examined LcAP2/ERF genes, including LcAP2/ERF-20, LcAP2/ERF-23, LcAP2/ERF-71, and LcAP2/ERF-107, suggested that the encoded TFs are likely involved in L. cylindrica’s response to cold stress. Recent research has shown that the overexpression of PtrERF108 can increase the viability of transgenic lemon (Citrus limon) in cold environments [40]. In rice, OsERF52 can directly regulate OsCBF1/2/3 expression and combine with OsERF52, OsICE1/OsbHLH002, or IPA1 in response to low-temperature stress [66]. In the current study, we found that the expression levels of some LcAP2/ERF genes were reduced or unchanged during the exposure to cold stress (e.g., LcERF-6, LcERF-45, LcERF-48, LcERF-64, LcERF-78, LcERF-113, LcERF-114, and LcERF-132). We consider that these LcAP2/ERF genes may be insensitive to low-temperature stress in luffa, but may be involved in the process of response to other biotic and/or abiotic stresses.

4.4. LcAP2/ERF Genes Associated with L. cylindrica Fruit Development

Members of the AP2/ERF subfamily also contribute to fruit development, for example, in Ficus carica [80], Solanum lycopersicum [82,93], and Actinidia deliciosa [94]. Recently, 146 ZbAP2/ERF genes were identified in Zanthoxylum bungeanum, many of which (e.g., ZbERF13.2, ZbERF3.1, and ZbERF2.1) were expressed at high levels in fruits at different developmental stages [10]. In apple, light and MeJA signals affect anthocyanin biosynthesis in developing fruits via the MdERF109–MdWER–MdJAZ2 module [95]. According to a recent study, SlERF.H5/H7 binds to the DRE cis-acting element in the promoter region of the cellulose synthase gene SlCESA3 to activate expression, thereby promoting the synthesis of cellulose to maintain cell wall integrity in developing tomato fruits [96]. However, in tomato, SlERF.F12 TF negatively regulates the onset of tomato fruit ripening by repressing the transcription of ripening-related genes through the transcriptional regulation of the co-repressor TOPLESS 2 and the histone deacetylases HDA1/HDA3 [93]. In the present study, at the primary stage of luffa fruit development, higher levels of LcAP2/ERF gene expression were found in male flowers, followed by the ovary, suggesting that the encoded LcAP2/ERF TFs play an active role during the early luffa fruit development period. Moreover, the expression levels of several LcAP2/ERF genes were significantly upregulated 2 days before flowering and 0, 2, 4, 6, 8, and 10 days after flowering, implying that the encoded LcAP2/ERF TFs have key functions throughout the luffa fruit development stage. In particular, the expression of LcERF-132 was upregulated nearly 90 times on day 4; however, this gene was expressed at very low levels throughout the low-temperature stress treatment period. In addition, the expression levels of a few LcAP2/ERF genes decreased as fruits developed (e.g., LcAP2/ERF-107, LcAP2/ERF-127, and LcAP2/ERF-128). Considering the functional diversity among AP2/ERF TFs, we speculate that these genes may contribute to L. cylindrica’s responses to other biological processes [3,14,66,67,84].

5. Conclusions

In this study, based on RNA-seq and RT-qPCR data from luffa, the expression levels 133 LcAP2/ERF genes were differentially affected under low-temperature stress conditions and during fruit development. In-depth analyses of the distribution locations on the LcAP2/ERF chromosomes, gene promoters and their structures, and protein evolutionary trees, as well as protein conserved structures and expression patterns, were carried out. According to the differential tissue-specific LcAP2/ERF gene expression profiles, as well as the expression patterns of low-temperature-treated and developing fruits, we identified several LcAP2/ERF genes potentially associated with the regulation of the cold stress response (e.g., LcAP2/ERF-20, LcAP2/ERF-23, LcAP2/ERF-71, and LcAP2/ERF-107) and fruit development (e.g., LcAP2/ERF-6, LcAP2/ERF-23, LcERF-64, and LcAP2/ERF-132) in L. cylindrica.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14112509/s1. File S1: Figure S1. Effects of low-temperature stress on 2-week-old luffa seedlings; File S2: Figure S2. L. cylindrica fruit phenotypes on different days before and after flowering; File S3: Table S1. Primers used in this study; File S4: Table S2. AP2/ERF family genes identified in luffa; File S5: Full-length sequences of 133 LcAP2/ERF genes; File S6: AP2/ERF protein sequences; File S7: Table S3. Duplicated LcAP2/ERF genes in L. cylindrica; File S8: Promoter region 2.0 kb upstream of LcAP2/ERF gene sequences; File S9: Identified cis-acting elements in LcAP2/ERF promoter regions; File S10: Table S4. Consistent sequences in the predicted AP2/ERF motifs in L. cylindrica; File S11: Tissue-specific LcAP2/ERF expression profiles; File S12: Low-temperature stress-induced LcAP2/ERF expression profiles.

Author Contributions

J.L.: writing—original draft, investigation, data curation, and methodology. H.Z. (Haifeng Zhong): data curation, resources, and conceptualization. C.C.: software, investigation, and data curation. Y.W.: formal analysis and data curation. Q.Z.: conceptualization and resources. Q.W.: writing—review and editing, and conceptualization. H.Z. (Haisheng Zhu): conceptualization, and writing—review and editing. Z.L.: project administration and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This study is supported by the Public Welfare Programme of Fujian Province (2024R1030003), the Fujian Academy of Agricultural Sciences Cooperation With Overseas Partners Program (DWHZ-2024-15), the National Vegetable Industry System for Bulk Vegetables (Fuzhou Comprehensive Experimental Station) (CARS-23-G51), and the ‘5511’ Chinese Academy of Agricultural Sciences-Fujian Academy of Agricultural Sciences Collaborative Innovation (XTCXGC2021003).

Data Availability Statement

All the transcriptome sequencing data used in this study can be download from the National Center for Biotechnology Information (NCBI) database (Genbank accession number: PRJNA1044273).

Acknowledgments

We are grateful to the editors and anonymous reviewers for their comments and suggestions for valuable revisions that improved this manuscript. We are grateful to the luffa research group at the Vegetable Research Institute of the Guangdong Academy of Agricultural Sciences for providing the luffa genome annotation dataset.

Conflicts of Interest

The authors have no conflicts of interest to declare.

References

  1. Wang, J.; Ren, Y.; Liu, X.; Luo, S.; Zhang, X.; Liu, X.; Lin, Q.; Zhu, S.; Wan, H.; Yang, Y. Transcriptional activation and phosphorylation of OsCNGC9 confer enhanced chilling tolerance in rice. Mol. Plant 2021, 14, 315–329. [Google Scholar] [CrossRef] [PubMed]
  2. Qian, Z.; Rao, X.; Zhang, R.; Gu, S.; Shen, Q.; Wu, H.; Lv, S.; Xie, L.; Li, X.; Wang, X. Genome-Wide Identification, Evolution, and Expression Analyses of AP2/ERF Family Transcription Factors in Erianthus fulvus. IJMS 2023, 24, 7102. [Google Scholar] [CrossRef] [PubMed]
  3. Xie, Z.; Nolan, T.M.; Jiang, H.; Yin, Y. AP2/ERF Transcription Factor Regulatory Networks in Hormone and Abiotic Stress Responses in Arabidopsis. Front. Plant Sci. 2019, 10, 228. [Google Scholar] [CrossRef] [PubMed]
  4. Zhang, J.; Liao, J.; Ling, Q.; Xi, Y.; Qian, Y. Genome-wide identification and expression profiling analysis of maize AP2/ERF superfamily genes reveal essential roles in abiotic stress tolerance. BMC Genom. 2022, 23, 125. [Google Scholar] [CrossRef] [PubMed]
  5. Cai, X.; Chen, Y.; Wang, Y.; Shen, Y.; Yang, J.; Jia, B.; Sun, X.; Sun, M. A comprehensive investigation of the regulatory roles of OsERF096, an AP2/ERF transcription factor, in rice cold stress response. Plant Cell Rep. 2023, 42, 2011–2022. [Google Scholar] [CrossRef]
  6. Mantiri, F.R.; Kurdyukov, S.; Lohar, D.P.; Sharopova, N.; Saeed, N.A.; Wang, X.D.; Vandenbosch, K.A.; Rose, R.J. The transcription factor MtSERF1 of the ERF subfamily identified by transcriptional profiling is required for somatic embryogenesis induced by auxin plus cytokinin in Medicago truncatula. Plant Physiol. 2008, 146, 1622–1636. [Google Scholar] [CrossRef]
  7. Sakuma, Y.; Liu, Q.; Dubouzet, J.G.; Abe, H.; Shinozaki, K.; Yamaguchi-Shinozaki, K. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochem. Biophys. Res. Commun. 2002, 290, 998–1009. [Google Scholar] [CrossRef]
  8. Kagaya, Y.; Ohmiya, K.; Hattori, T. RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants. Nucleic Acids Res. 1999, 27, 470–478. [Google Scholar] [CrossRef]
  9. Je, B.I.; Piao, H.L.; Park, S.J.; Park, S.H.; Kim, C.M.; Xuan, Y.H.; Park, S.H.; Huang, J.; Do Choi, Y.; An, G.; et al. RAV-Like1 maintains brassinosteroid homeostasis via the coordinated activation of BRI1 and biosynthetic genes in rice. Plant Cell 2010, 22, 1777–1791. [Google Scholar] [CrossRef]
  10. Ma, L.; Shi, Q.; Ma, Q.; Wang, X.; Chen, X.; Han, P.; Luo, Y.; Hu, H.; Fei, X.; Wei, A. Genome-wide analysis of AP2/ERF transcription factors that regulate fruit development of Chinese prickly ash. BMC Plant Biol. 2024, 24, 565. [Google Scholar] [CrossRef]
  11. Qi, X.; Liu, L.; Liu, C.; Song, L.; Dong, Y.; Chen, L.; Li, M. Sweet cherry AP2/ERF transcription factor, PavRAV2, negatively modulates fruit size by directly repressing PavKLUH expression. Physiol. Plant 2023, 175, e14065. [Google Scholar] [CrossRef] [PubMed]
  12. Cheng, C.; An, L.; Li, F.; Ahmad, W.; Aslam, M.; Ul Haq, M.Z.; Yan, Y.; Ahmad, R.M. Wide-Range Portrayal of AP2/ERF Transcription Factor Family in Maize (Zea mays L.) Development and Stress Responses. Genes 2023, 14, 194. [Google Scholar] [CrossRef]
  13. Dong, L.; Cheng, Y.; Wu, J.; Cheng, Q.; Li, W.; Fan, S.; Jiang, L.; Xu, Z.; Kong, F.; Zhang, D.; et al. Overexpression of GmERF5, a new member of the soybean EAR motif-containing ERF transcription factor, enhances resistance to Phytophthora sojae in soybean. J. Exp. Bot. 2015, 66, 2635–2647. [Google Scholar] [CrossRef]
  14. Zhao, Y.; Wei, T.; Yin, K.Q.; Chen, Z.; Gu, H.; Qu, L.J.; Qin, G. Arabidopsis RAP2.2 plays an important role in plant resistance to Botrytis cinerea and ethylene responses. New Phytol. 2012, 195, 450–460. [Google Scholar] [CrossRef] [PubMed]
  15. Chen, N.; Qin, J.; Tong, S.; Wang, W.; Jiang, Y. One AP2/ERF Transcription Factor Positively Regulates Pi Uptake and Drought Tolerance in Poplar. IJMS 2022, 23, 5241. [Google Scholar] [CrossRef]
  16. Chen, K.; Tang, W.; Zhou, Y.; Chen, J.; Xu, Z.; Ma, R.; Dong, Y.; Ma, Y.; Chen, M. AP2/ERF transcription factor GmDREB1 confers drought tolerance in transgenic soybean by interacting with GmERFs. Plant Physiol. Biochem. 2022, 170, 287–295. [Google Scholar] [CrossRef]
  17. Kong, L.; Song, Q.; Wei, H.; Wang, Y.; Lin, M.; Sun, K.; Zhang, Y.; Yang, J.; Li, C.; Luo, K. The AP2/ERF transcription factor PtoERF15 confers drought tolerance via JA-mediated signaling in Populus. New Phytol. 2023, 240, 1848–1867. [Google Scholar] [CrossRef] [PubMed]
  18. Zhang, J.; Shi, S.Z.; Jiang, Y.; Zhong, F.; Liu, G.; Yu, C.; Lian, B.; Chen, Y. Genome-wide investigation of the AP2/ERF superfamily and their expression under salt stress in Chinese willow (Salix matsudana). PeerJ 2021, 9, e11076. [Google Scholar] [CrossRef]
  19. Magar, M.M.; Liu, H.; Yan, G. Genome-Wide Analysis of AP2/ERF Superfamily Genes in Contrasting Wheat Genotypes Reveals Heat Stress-Related Candidate Genes. Front. Plant Sci. 2022, 13, 853086. [Google Scholar] [CrossRef]
  20. Zhao, M.; Li, Y.; Zhang, X.; You, X.; Yu, H.; Guo, R.; Zhao, X. Genome-Wide Identification of AP2/ERF Superfamily Genes in Juglans mandshurica and Expression Analysis under Cold Stress. IJMS 2022, 23, 15225. [Google Scholar] [CrossRef]
  21. Du, C.; Hu, K.; Xian, S.; Liu, C.; Fan, J.; Tu, J.; Fu, T. Dynamic transcriptome analysis reveals AP2/ERF transcription factors responsible for cold stress in rapeseed (Brassica napus L.). Mol. Genet. Genomics 2016, 291, 1053–1067. [Google Scholar] [CrossRef] [PubMed]
  22. Xie, W.; Ding, C.; Hu, H.; Dong, G.; Zhang, G.; Qian, Q.; Ren, D. Molecular Events of Rice AP2/ERF Transcription Factors. IJMS 2022, 23, 12013. [Google Scholar] [CrossRef] [PubMed]
  23. Zhang, L.; Chen, L.; Pang, S.; Zheng, Q.; Quan, S.; Liu, Y.; Xu, T.; Liu, Y.; Qi, M. Function Analysis of the ERF and DREB Subfamilies in Tomato Fruit Development and Ripening. Front. Plant Sci. 2022, 13, 849048. [Google Scholar] [CrossRef]
  24. Hu, L.; Liu, S. Genome-wide identification and phylogenetic analysis of the ERF gene family in cucumbers. Genet. Mol. Biol. 2011, 34, 624–634. [Google Scholar] [CrossRef]
  25. Wan, R.; Song, J.; Lv, Z.; Qi, X.; Han, X.; Guo, Q.; Wang, S.; Shi, J.; Jian, Z.; Hu, Q.; et al. Genome-Wide Identification and Comprehensive Analysis of the AP2/ERF Gene Family in Pomegranate Fruit Development and Postharvest Preservation. Genes 2022, 13, 895. [Google Scholar] [CrossRef]
  26. Zhang, Z.; Li, X. Genome-wide identification of AP2/ERF superfamily genes and their expression during fruit ripening of Chinese jujube. Sci. Rep. 2018, 8, 15612. [Google Scholar] [CrossRef] [PubMed]
  27. Zhang, Y.; Guo, C.; Deng, M.; Li, S.; Chen, Y.; Gu, X.; Tang, G.; Lin, Y.; Wang, Y.; He, W.; et al. Genome-Wide Analysis of the ERF Family and Identification of Potential Genes Involved in Fruit Ripening in Octoploid Strawberry. IJMS 2022, 23, 10550. [Google Scholar] [CrossRef]
  28. Shi, Y.; Ding, Y.; Yang, S. Molecular regulation of CBF signaling in cold acclimation. Trends Plant Sci. 2018, 23, 623–637. [Google Scholar] [CrossRef]
  29. Meng, D.; Li, S.; Feng, X.; Di, Q.; Zhou, M.; Yu, X.; He, C.; Yan, Y.; Wang, J.; Sun, M.; et al. CsBPC2 is essential for cucumber survival under cold stress. BMC Plant Biol. 2023, 23, 566. [Google Scholar] [CrossRef]
  30. Cheng, X.; Qin, M.; Chen, R.; Jia, Y.; Zhu, Q.; Chen, G.; Wang, A.; Ling, B.; Rong, W. Citrullus colocynthis (L.) schrad.: A promising pharmaceutical resource for multiple diseases. Molecules 2023, 28, 6221. [Google Scholar] [CrossRef]
  31. Tang, L.; He, Y.; Liu, B.; Xu, Y.; Zhao, G. Genome-wide identification and characterization analysis of WUSCHEL-related homeobox family in melon (Cucumis melo L.). IJMS 2023, 24, 12326. [Google Scholar] [CrossRef] [PubMed]
  32. Li, H.; Kong, F.; Tang, T.; Luo, Y.; Gao, H.; Xu, J.; Xing, G.; Li, L. Physiological and transcriptomic analyses revealed that humic acids improve low-temperature stress tolerance in zucchini (Cucurbita pepo L.) Seedlings. Plants 2023, 12, 548. [Google Scholar] [CrossRef] [PubMed]
  33. Liu, J.; Peng, L.; Cao, C.; Bai, C.; Wang, Y.; Li, Z.; Zhu, H.; Wen, Q.; He, S. Identification of WRKY family members and characterization of the low-temperature-stress-responsive WRKY genes in Luffa (Luffa cylindrica L.). Plants 2024, 13, 676. [Google Scholar] [CrossRef]
  34. Feng, K.; Hou, X.; Xing, G.; Liu, J.; Duan, A.; Xu, Z.; Li, M.; Zhuang, J.; Xiong, A. Advances in AP2/ERF super-family transcription factors in plant. Crit. Rev. Biotechnol. 2020, 40, 750–776. [Google Scholar] [CrossRef] [PubMed]
  35. Ma, Z.; Hu, L.; Jiang, W. Understanding AP2/ERF Transcription Factor Responses and Tolerance to Various Abiotic Stresses in Plants: A Comprehensive Review. IJMS 2024, 25, 893. [Google Scholar] [CrossRef]
  36. Miura, K.; Tada, Y. Regulation of water, salinity, and cold stress responses by salicylic acid. Front. Plant Sci. 2014, 5, 4. [Google Scholar] [CrossRef]
  37. Xie, Z.; Yang, C.; Liu, S.; Li, M.; Gu, L.; Peng, X.; Zhang, Z. Identification of AP2/ERF transcription factors in Tetrastigma hemsleyanum revealed the specific roles of ERF46 under cold stress. Front. Plant Sci. 2022, 13, 936602. [Google Scholar] [CrossRef]
  38. Yin, F.; Zeng, Y.; Ji, J.; Wang, P.; Zhang, Y.; Li, W. The Halophyte Halostachys caspica AP2/ERF Transcription Factor HcTOE3 Positively Regulates Freezing Tolerance in Arabidopsis. Front. Plant Sci. 2021, 12, 638788. [Google Scholar] [CrossRef]
  39. Sun, X.; Zhang, L.; Wong, D.C.J.; Wang, Y.; Zhu, Z.; Xu, G.; Wang, Q.; Li, S.; Liang, Z.; Xin, H. The ethylene response factor VaERF092 from Amur grape regulates the transcription factor VaWRKY33, improving cold tolerance. Plant J. 2019, 99, 988–1002. [Google Scholar] [CrossRef]
  40. Khan, M.; Hu, J.; Dahro, B.; Ming, R.; Zhang, Y.; Wang, Y.; Alhag, A.; Li, C.; Liu, J.H. ERF108 from Poncirus trifoliata (L.) Raf. functions in cold tolerance by modulating raffinose synthesis through transcriptional regulation of PtrRafS. Plant J. 2021, 108, 705–724. [Google Scholar] [CrossRef]
  41. Yuste-Lisbona, F.J.; Fernández-Lozano, A.; Pineda, B.; Bretones, S.; Ortíz-Atienza, A.; García-Sogo, B.; Müller, N.A.; Angosto, T.; Capel, J.; Moreno, V.; et al. ENO regulates tomato fruit size through the floral meristem development network. Proc. Natl. Acad. Sci. USA 2020, 117, 8187–8195. [Google Scholar] [CrossRef] [PubMed]
  42. Abdel-Hamid, H.; Chin, K.; Moeder, W.; Yoshioka, K. High throughput chemical screening supports the involvement of Ca2+ in cyclic nucleotide-gated ion channel-mediated programmed cell death in Arabidopsis. Plant Signal Behav. 2011, 6, 1817–1819. [Google Scholar] [CrossRef] [PubMed]
  43. Lee, S.K.; Lee, S.M.; Kim, M.H.; Park, S.K.; Jung, K.H. Genome-Wide Analysis of Cyclic Nucleotide-Gated Channel Genes Related to Pollen Development in Rice. Plants 2022, 11, 3145. [Google Scholar] [CrossRef]
  44. Hao, L.; Qiao, X. Genome-wide identification and analysis of the CNGC gene family in maize. PeerJ 2018, 6, e5816. [Google Scholar] [CrossRef]
  45. Saand, M.A.; Xu, Y.P.; Munyampundu, J.P.; Li, W.; Zhang, X.R.; Cai, X.Z. Phylogeny and evolution of plant cyclic nucleotide-gated ion channel (CNGC) gene family and functional analyses of tomato CNGCs. DNA Res. 2015, 22, 471–483. [Google Scholar] [CrossRef]
  46. Zhang, N.; Lin, H.; Zeng, Q.; Fu, D.; Gao, X.; Wu, J.; Feng, X.; Wang, Q.; Ling, Q.; Wu, Z. Genome-wide identification and expression analysis of the cyclic nucleotide-gated ion channel (CNGC) gene family in Saccharum spontaneum. BMC Genom. 2023, 24, 281. [Google Scholar] [CrossRef] [PubMed]
  47. Jiang, Z.; Du, L.; Shen, L.; He, J.; Xia, X.; Zhang, L.; Yang, X. Genome-Wide Exploration and Expression Analysis of the CNGC Gene Family in Eggplant (Solanum melongena L.) under Cold Stress, with Functional Characterization of SmCNGC1a. IJMS 2023, 24, 13049. [Google Scholar] [CrossRef]
  48. Baloch, A.A.; Raza, A.M.; Rana, S.S.A.; Ullah, S.; Khan, S.; Zaib Un, N.; Zahid, H.; Malghani, G.K.; Kakar, K.U. BrCNGC gene family in field mustard: Genome-wide identification, characterization, comparative synteny, evolution and expression profiling. Sci. Rep. 2021, 11, 24203. [Google Scholar] [CrossRef]
  49. Wang, L.; Li, M.; Liu, Z.; Dai, L.; Zhang, M.; Wang, L.; Zhao, J.; Liu, M. Genome-wide identification of CNGC genes in Chinese jujube (Ziziphus jujuba Mill.) and ZjCNGC2 mediated signalling cascades in response to cold stress. BMC Genom. 2020, 21, 191. [Google Scholar] [CrossRef]
  50. Chen, L.; Wang, W.; He, H.; Yang, P.; Sun, X.; Zhang, Z. Genome-Wide Identification, Characterization and Experimental Expression Analysis of CNGC Gene Family in Gossypium. IJMS 2023, 24, 4617. [Google Scholar] [CrossRef]
  51. Mao, X.; Wang, C.; Lv, Q.; Tian, Y.; Wang, D.; Chen, B.; Mao, J.; Li, W.; Chu, M.; Zuo, C. Cyclic nucleotide gated channel genes (CNGCs) in Rosaceae: Genome-wide annotation, evolution and the roles on Valsa canker resistance. Plant Cell Rep. 2021, 40, 2369–2382. [Google Scholar] [CrossRef] [PubMed]
  52. Xie, D.; Xu, Y.; Wang, J.; Liu, W.; Zhou, Q.; Luo, S.; Huang, W.; He, X.; Li, Q.; Peng, Q.; et al. The wax gourd genomes offer insights into the genetic diversity and ancestral cucurbit karyotype. Nat. Commun. 2019, 10, 5158. [Google Scholar] [CrossRef] [PubMed]
  53. Guo, S.; Zhang, J.; Sun, H.; Salse, J.; Lucas, W.J.; Zhang, H.; Zheng, Y.; Mao, L.; Ren, Y.; Wang, Z. The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions. Nat. Genet. 2013, 45, 51–58. [Google Scholar] [CrossRef]
  54. Sun, H.; Wu, S.; Zhang, G.; Jiao, C.; Guo, S.; Ren, Y.; Zhang, J.; Zhang, H.; Gong, G.; Jia, Z. Karyotype stability and unbiased fractionation in the paleo-allotetraploid Cucurbita genomes. Mol. Plant 2017, 10, 1293–1306. [Google Scholar] [CrossRef]
  55. Garcia-Mas, J.; Benjak, A.; Sanseverino, W.; Bourgeois, M.; Mir, G.; González, V.M.; Hénaff, E.; Câmara, F.; Cozzuto, L.; Lowy, E. The genome of melon (Cucumis melo L.). Proc. Natl. Acad. Sci. USA 2012, 109, 11872–11877. [Google Scholar] [CrossRef]
  56. Montero-Pau, J.; Blanca, J.; Bombarely, A.; Ziarsolo, P.; Esteras, C.; Martí-Gómez, C.; Ferriol, M.; Gómez, P.; Jamilena, M.; Mueller, L. De novo assembly of the zucchini genome reveals a whole-genome duplication associated with the origin of the Cucurbita genus. Plant Biotechnol. J. 2018, 16, 1161–1171. [Google Scholar] [CrossRef]
  57. Wu, H.; Zhao, G.; Gong, H.; Li, J.; Luo, C.; He, X.; Luo, S.; Zheng, X.; Liu, X.; Guo, J.; et al. A high-quality sponge gourd (Luffa cylindrica) genome. Hortic. Res. 2020, 7, 128. [Google Scholar] [CrossRef] [PubMed]
  58. Liu, J.; Wang, B.; Li, Y.; Huang, L.; Zhang, Q.; Zhu, H.; Wen, Q. RNA sequencing analysis of low temperature and low light intensity-responsive transcriptomes of zucchini (Cucurbita pepo L.). Sci. Hortic. 2020, 265, 109263. [Google Scholar] [CrossRef]
  59. Langdon, W.B. Performance of genetic programming optimised Bowtie2 on genome comparison and analytic testing (GCAT) benchmarks. BioData Min. 2015, 8, 1. [Google Scholar] [CrossRef]
  60. Zhao, Y.; Li, M.C.; Konaté, M.M.; Chen, L.; Das, B.; Karlovich, C.; Williams, P.M.; Evrard, Y.A.; Doroshow, J.H.; McShane, L.M. TPM, FPKM, or normalized counts a comparative study of quantification measures for the analysis of RNA-seq data from the NCI patient-derived models repository. J. Transl. Med. 2021, 19, 269. [Google Scholar] [CrossRef]
  61. Liu, J.; Wang, Y.; Ye, X.; Zhang, Q.; Li, Y.; Chen, M.; Wang, B.; Bai, C.; Li, Z.; Wen, Q.; et al. Genome-wide identification and expression analysis of the WRKY gene family in response to low-temperature and drought stresses in Cucurbita pepo L. Sci. Hortic. 2024, 330, 113048. [Google Scholar] [CrossRef]
  62. Wang, D.; Zhang, Y.; Zhang, Z.; Zhu, J.; Yu, J. KaKs_Calculator 2.0: A toolkit incorporating gamma-series methods and sliding window strategies. Genom. Proteom. Bioinform. 2010, 8, 77–80. [Google Scholar] [CrossRef]
  63. Castellanos, M.D.P.; Wickramasinghe, C.D.; Betrán, E. The roles of gene duplications in the dynamics of evolutionary conflicts. Proc. Biol. Sci. 2024, 291, 20240555. [Google Scholar] [CrossRef] [PubMed]
  64. Dorshorst, B.; Harun-Or-Rashid, M.; Bagherpoor, A.J.; Rubin, C.J.; Ashwell, C.; Gourichon, D.; Tixier-Boichard, M.; Hallböök, F.; Andersson, L. A genomic duplication is associated with ectopic eomesodermin expression in the embryonic chicken comb and two duplex-comb phenotypes. PLoS Genet. 2015, 11, e1004947. [Google Scholar] [CrossRef] [PubMed]
  65. Hu, F.; Zhang, Y.; Guo, J. Identification and Characterization of AP2/ERF Transcription Factors in Yellow Horn. IJMS 2022, 23, 14991. [Google Scholar] [CrossRef]
  66. Xu, L.; Yang, L.; Li, A.; Guo, J.; Wang, H.; Qi, H.; Li, M.; Yang, P.; Song, S. An AP2/ERF transcription factor confers chilling tolerance in rice. Sci. Adv. 2024, 10, eado4788. [Google Scholar] [CrossRef]
  67. Zhu, J.; Wei, X.; Yin, C.; Zhou, H.; Yan, J.; He, W.; Yan, J.; Li, H. ZmEREB57 regulates OPDA synthesis and enhances salt stress tolerance through two distinct signalling pathways in Zea mays. Plant Cell Environ. 2023, 46, 2867–2883. [Google Scholar] [CrossRef]
  68. Nakano, T.; Suzuki, K.; Fujimura, T.; Shinshi, H. Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol. 2006, 140, 411–432. [Google Scholar] [CrossRef]
  69. Li, D.; Liu, X.; Shu, L.; Zhang, H.; Zhang, S.; Song, Y.; Zhang, Z. Global analysis of the AP2/ERF gene family in rose (Rosa chinensis) genome unveils the role of RcERF099 in Botrytis resistance. BMC Plant Biol. 2020, 20, 533. [Google Scholar] [CrossRef]
  70. Guo, J.; Xu, W.; Hu, Y.; Huang, J.; Zhao, Y.; Zhang, L.; Huang, C.; Ma, H. Phylotranscriptomics in Cucurbitaceae reveal multiple whole-genome duplications and key morphological and molecular innovations. Mol. Plant 2020, 13, 1117–1133. [Google Scholar] [CrossRef]
  71. Barrera-Redondo, J.; Lira-Saade, R.; Eguiarte, L.E. Gourds and tendrils of cucurbitaceae: How their shape diversity, molecular and morphological novelties evolved via whole-genome duplications. Mol. Plant 2020, 13, 1108–1110. [Google Scholar] [CrossRef] [PubMed]
  72. Zhuang, J.; Cai, B.; Peng, R.H.; Zhu, B.; Jin, X.F.; Xue, Y.; Gao, F.; Fu, X.Y.; Tian, Y.S.; Zhao, W.; et al. Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa. Biochem. Biophys. Res. Commun. 2008, 371, 468–474. [Google Scholar] [CrossRef] [PubMed]
  73. Licausi, F.; Giorgi, F.M.; Zenoni, S.; Osti, F.; Pezzotti, M.; Perata, P. Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera. BMC Genom. 2010, 11, 719. [Google Scholar] [CrossRef] [PubMed]
  74. Shigyo, M.; Ito, M. Analysis of gymnosperm two-AP2-domain-containing genes. Dev. Genes. Evol. 2004, 214, 105–114. [Google Scholar] [CrossRef] [PubMed]
  75. Ritonga, F.N.; Ngatia, J.N.; Wang, Y.; Khoso, M.A.; Farooq, U.; Chen, S. AP2/ERF, an important cold stress-related transcription factor family in plants: A review. Physiol. Mol. Biol. Plants 2021, 27, 1953–1968. [Google Scholar] [CrossRef]
  76. Matías-Hernández, L.; Aguilar-Jaramillo, A.E.; Marín-González, E.; Suárez-López, P.; Pelaz, S. RAV genes: Regulation of floral induction and beyond. Ann. Bot. 2014, 114, 1459–1470. [Google Scholar] [CrossRef]
  77. An, J.P.; Xu, R.R.; Liu, X.; Su, L.; Yang, K.; Wang, X.F.; Wang, G.L.; You, C.X. Abscisic acid insensitive 4 interacts with ICE1 and JAZ proteins to regulate ABA signaling-mediated cold tolerance in apple. J. Exp. Bot. 2022, 73, 980–997. [Google Scholar] [CrossRef]
  78. Wu, L.; Chen, X.; Ren, H.; Zhang, Z.; Zhang, H.; Wang, J.; Wang, X.C.; Huang, R. ERF protein JERF1 that transcriptionally modulates the expression of abscisic acid biosynthesis-related gene enhances the tolerance under salinity and cold in tobacco. Planta 2007, 226, 815–825. [Google Scholar] [CrossRef]
  79. Agurla, S.; Gahir, S.; Munemasa, S.; Murata, Y.; Raghavendra, A.S. Mechanism of Stomatal Closure in Plants Exposed to Drought and Cold Stress. Adv. Exp. Med. Biol. 2018, 1081, 215–232. [Google Scholar] [CrossRef]
  80. Cui, Y.; Zhai, Y.; He, J.; Song, M.; Flaishman, M.A.; Ma, H. AP2/ERF genes associated with superfast fig (Ficus carica L.) fruit ripening. Front. Plant Sci. 2022, 13, 1040796. [Google Scholar] [CrossRef]
  81. Zhang, Y.; Ming, R.; Khan, M.; Wang, Y.; Dahro, B.; Xiao, W.; Li, C.; Liu, J.H. ERF9 of Poncirus trifoliata (L.) Raf. undergoes feedback regulation by ethylene and modulates cold tolerance via regulating a glutathione S-transferase U17 gene. Plant Biotechnol. J. 2022, 20, 183–200. [Google Scholar] [CrossRef] [PubMed]
  82. Deng, H.; Pei, Y.; Xu, X.; Du, X.; Xue, Q.; Gao, Z.; Shu, P.; Wu, Y.; Liu, Z.; Jian, Y.; et al. Ethylene-MPK8-ERF.C1-PR module confers resistance against Botrytis cinerea in tomato fruit without compromising ripening. New Phytol. 2024, 242, 592–609. [Google Scholar] [CrossRef] [PubMed]
  83. Tournier, B.; Sanchez-Ballesta, M.T.; Jones, B.; Pesquet, E.; Regad, F.; Latché, A.; Pech, J.C.; Bouzayen, M. New members of the tomato ERF family show specific expression pattern and diverse DNA-binding capacity to the GCC box element. FEBS Lett. 2003, 550, 149–154. [Google Scholar] [CrossRef] [PubMed]
  84. Wang, M.; Gao, M.; Zhao, Y.; Chen, Y.; Wu, L.; Yin, H.; Xiong, S.; Wang, S.; Wang, J.; Yang, Y.; et al. LcERF19, an AP2/ERF transcription factor from Litsea cubeba, positively regulates geranial and neral biosynthesis. Hortic. Res. 2022, 9, uhac093. [Google Scholar] [CrossRef]
  85. Van der Does, D.; Leon-Reyes, A.; Koornneef, A.; Van Verk, M.C.; Rodenburg, N.; Pauwels, L.; Goossens, A.; Körbes, A.P.; Memelink, J.; Ritsema, T.; et al. Salicylic acid suppresses jasmonic acid signaling downstream of SCFCOI1-JAZ by targeting GCC promoter motifs via transcription factor ORA59. Plant Cell 2013, 25, 744–761. [Google Scholar] [CrossRef]
  86. Caarls, L.; Van der Does, D.; Hickman, R.; Jansen, W.; Verk, M.C.; Proietti, S.; Lorenzo, O.; Solano, R.; Pieterse, C.M.; Van Wees, S.C. Assessing the Role of ETHYLENE RESPONSE FACTOR Transcriptional Repressors in Salicylic Acid-Mediated Suppression of Jasmonic Acid-Responsive Genes. Plant Cell Physiol. 2017, 58, 266–278. [Google Scholar] [CrossRef]
  87. Lv, B.; Wei, K.; Hu, K.; Tian, T.; Zhang, F.; Yu, Z.; Zhang, D.; Su, Y.; Sang, Y.; Zhang, X.; et al. MPK14-mediated auxin signaling controls lateral root development via ERF13-regulated very-long-chain fatty acid biosynthesis. Mol. Plant 2021, 14, 285–297. [Google Scholar] [CrossRef]
  88. Gambhir, P.; Singh, V.; Parida, A.; Raghuvanshi, U.; Kumar, R.; Sharma, A.K. Ethylene response factor ERF.D7 activates auxin response factor 2 paralogs to regulate tomato fruit ripening. Plant Physiol. 2022, 190, 2775–2796. [Google Scholar] [CrossRef]
  89. Zhou, S.M.; Wang, F.; Yan, S.Y.; Zhu, Z.M.; Gao, X.F.; Zhao, X.L. Phylogenomics and plastome evolution of Indigofera (Fabaceae). Front. Plant Sci. 2023, 14, 1186598. [Google Scholar] [CrossRef]
  90. Guo, Z.; Cai, L.; Liu, C.; Chen, Z.; Guan, S.; Ma, W.; Pan, G. Low-temperature stress affects reactive oxygen species, osmotic adjustment substances, and antioxidants in rice (Oryza sativa L.) at the reproductive stage. Sci. Rep. 2022, 12, 6224. [Google Scholar] [CrossRef]
  91. Fei, J.; Wang, Y.S.; Cheng, H.; Su, Y.B.; Zhong, Y.J.; Zheng, L. The Kandelia obovata transcription factor KoWRKY40 enhances cold tolerance in transgenic Arabidopsis. BMC Plant Biol. 2022, 22, 274. [Google Scholar] [CrossRef] [PubMed]
  92. Lang, T.; Tang, Y.; Tam, N.F.; Gan, K.; Wu, J.; Wu, W.; Fu, Y.; Li, M.; Hu, Z.; Li, F.; et al. Microcosm study on cold adaptation and recovery of an exotic mangrove plant, Laguncularia racemosa in China. Mar. Environ. Res. 2022, 176, 105611. [Google Scholar] [CrossRef] [PubMed]
  93. Deng, H.; Chen, Y.; Liu, Z.; Liu, Z.; Shu, P.; Wang, R.; Hao, Y.; Su, D.; Pirrello, J.; Liu, Y.; et al. SlERF.F12 modulates the transition to ripening in tomato fruit by recruiting the co-repressor TOPLESS and histone deacetylases to repress key ripening genes. Plant Cell 2022, 34, 1250–1272. [Google Scholar] [CrossRef] [PubMed]
  94. Yin, X.R.; Allan, A.C.; Chen, K.S.; Ferguson, I.B. Kiwifruit EIL and ERF genes involved in regulating fruit ripening. Plant Physiol. 2010, 153, 1280–1292. [Google Scholar] [CrossRef]
  95. Zhang, X.; Yu, L.; Zhang, M.; Wu, T.; Song, T.; Yao, Y.; Zhang, J.; Tian, J. MdWER interacts with MdERF109 and MdJAZ2 to mediate methyl jasmonate- and light-induced anthocyanin biosynthesis in apple fruit. Plant J. 2024, 118, 1327–1342. [Google Scholar] [CrossRef]
  96. Pei, Y.; Xue, Q.; Shu, P.; Xu, W.; Du, X.; Wu, M.; Liu, K.; Pirrello, J.; Bouzayen, M.; Hong, Y.; et al. Bifunctional transcription factors SlERF.H5 and H7 activate cell wall and repress gibberellin biosynthesis genes in tomato via a conserved motif. Dev. Cell 2024, 59, 1345–1359.e6. [Google Scholar] [CrossRef]
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Figure 1. The chromosomal distribution of the LcAP2/ERF gene in luffa. The location of the chromosome for each LcAP2/ERF gene can be identified by the scale on the left. Tandemly and segmentally duplicated genes are marked in red and blue, respectively.
Figure 1. The chromosomal distribution of the LcAP2/ERF gene in luffa. The location of the chromosome for each LcAP2/ERF gene can be identified by the scale on the left. Tandemly and segmentally duplicated genes are marked in red and blue, respectively.
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Figure 2. Analyses of LcAP2/ERF promoter elements and structure. (a) The PlantCARE database and TBtools software (version 1.6) were used to analyze LcAP2/ERF gene promoter elements. (b) LcAP2/ERF gene structures were constructed using Gene Structure Display Server (GSDS) software (version 2.0).
Figure 2. Analyses of LcAP2/ERF promoter elements and structure. (a) The PlantCARE database and TBtools software (version 1.6) were used to analyze LcAP2/ERF gene promoter elements. (b) LcAP2/ERF gene structures were constructed using Gene Structure Display Server (GSDS) software (version 2.0).
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Figure 3. Phylogenetic relationships and analysis of conserved structural domains of LcAP2/ERF proteins in luffa. (a) The protein phylogenetic tree was constructed using MEGA (version 7.0), based on the 133 full-length LcAP2/ERF protein sequences in luffa. (b) Analysis of the distribution of 16 conserved motifs in LcAP2/ERF proteins.
Figure 3. Phylogenetic relationships and analysis of conserved structural domains of LcAP2/ERF proteins in luffa. (a) The protein phylogenetic tree was constructed using MEGA (version 7.0), based on the 133 full-length LcAP2/ERF protein sequences in luffa. (b) Analysis of the distribution of 16 conserved motifs in LcAP2/ERF proteins.
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Figure 4. Heatmap of seven different tissue-expression-specific and low-temperature-stress-induced LcAP2/ERF gene expressions in luffa. Expression heatmap of the LcAP2/ERF genes was drawn based on published RNA-seq data. Color coding indicates normalized log10-converted FPKM values, while red and blue indicate high and low expression levels, respectively. (a) Tissue-specific luffa LcAP2/ERF expression levels. (b) Expression levels of LcAP2/ERF genes in luffa leaves at five time points under low-temperature treatment.
Figure 4. Heatmap of seven different tissue-expression-specific and low-temperature-stress-induced LcAP2/ERF gene expressions in luffa. Expression heatmap of the LcAP2/ERF genes was drawn based on published RNA-seq data. Color coding indicates normalized log10-converted FPKM values, while red and blue indicate high and low expression levels, respectively. (a) Tissue-specific luffa LcAP2/ERF expression levels. (b) Expression levels of LcAP2/ERF genes in luffa leaves at five time points under low-temperature treatment.
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Figure 5. RT-qPCR analyses of the expressions of 14 luffa LcAP2/ERF genes relevant to low-temperature sensitivity. The Lc18S rRNA gene expression levels were employed as a control to calculate all levels. And the error bars in the figure represent the standard errors of the three biological replicates.
Figure 5. RT-qPCR analyses of the expressions of 14 luffa LcAP2/ERF genes relevant to low-temperature sensitivity. The Lc18S rRNA gene expression levels were employed as a control to calculate all levels. And the error bars in the figure represent the standard errors of the three biological replicates.
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Figure 6. The regression analyses of the values of folding changes were established from RNA-seq and RT-qPCR data. Expression change fold was calculated in RNA-seq analyses by comparing FPKM values at 2, 4, 8, and 12 h to FPKM values at 0 h. Alternatively, when RT-qPCR analyses were performed, the expression levels at 2, 4, 8, and 12 h were normalized to the expression level at 0 h to calculate fold change.
Figure 6. The regression analyses of the values of folding changes were established from RNA-seq and RT-qPCR data. Expression change fold was calculated in RNA-seq analyses by comparing FPKM values at 2, 4, 8, and 12 h to FPKM values at 0 h. Alternatively, when RT-qPCR analyses were performed, the expression levels at 2, 4, 8, and 12 h were normalized to the expression level at 0 h to calculate fold change.
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Figure 7. Relative expression levels of 14 selected LcAP2/ERF genes associated with L. cylindrica fruit development. Note that −2 d represents 2 d before flowering, and 0, 2, 4, 6, 8, 10 d represent 0, 2, 4, 6, 8, 10 d after flowering, respectively. Lc18S rRNA gene expression levels were employed as a control to calculate all levels. And the error bars in the figure represent the standard errors of the three biological replicates.
Figure 7. Relative expression levels of 14 selected LcAP2/ERF genes associated with L. cylindrica fruit development. Note that −2 d represents 2 d before flowering, and 0, 2, 4, 6, 8, 10 d represent 0, 2, 4, 6, 8, 10 d after flowering, respectively. Lc18S rRNA gene expression levels were employed as a control to calculate all levels. And the error bars in the figure represent the standard errors of the three biological replicates.
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Figure 8. Subcellular localization of LcAP2/ERF proteins. (a) Construction of pCAMBIA1300-LcAP2/ERF-71-GFP and pCAMBIA1300-LcAP2/ERF-107-GFP recombinant vectors. (b) Bright field, DAPI, green fluorescence, and combined images of DAPI, green fluorescence, and bright field are shown. 35S::GFP indicates Gv3101 Agrobacterium tumefaciens strain carrying the empty vector (pCAMBIA1300-GFP); 35S::LcAP2/ERF-71::GFP and 35S::LcAP2/ERF-107::GFP represent A. tumefaciens strains carrying pCAMBIA1300-LcAP2/ERF-71-GFP and pCAMBIA1300-LcAP2/ERF-107-GFP, respectively. Scale bar = 50 µM.
Figure 8. Subcellular localization of LcAP2/ERF proteins. (a) Construction of pCAMBIA1300-LcAP2/ERF-71-GFP and pCAMBIA1300-LcAP2/ERF-107-GFP recombinant vectors. (b) Bright field, DAPI, green fluorescence, and combined images of DAPI, green fluorescence, and bright field are shown. 35S::GFP indicates Gv3101 Agrobacterium tumefaciens strain carrying the empty vector (pCAMBIA1300-GFP); 35S::LcAP2/ERF-71::GFP and 35S::LcAP2/ERF-107::GFP represent A. tumefaciens strains carrying pCAMBIA1300-LcAP2/ERF-71-GFP and pCAMBIA1300-LcAP2/ERF-107-GFP, respectively. Scale bar = 50 µM.
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Liu, J.; Zhong, H.; Cao, C.; Wang, Y.; Zhang, Q.; Wen, Q.; Zhu, H.; Li, Z. Identification of AP2/ERF Transcription Factors and Characterization of AP2/ERF Genes Related to Low-Temperature Stress Response and Fruit Development in Luffa. Agronomy 2024, 14, 2509. https://doi.org/10.3390/agronomy14112509

AMA Style

Liu J, Zhong H, Cao C, Wang Y, Zhang Q, Wen Q, Zhu H, Li Z. Identification of AP2/ERF Transcription Factors and Characterization of AP2/ERF Genes Related to Low-Temperature Stress Response and Fruit Development in Luffa. Agronomy. 2024; 14(11):2509. https://doi.org/10.3390/agronomy14112509

Chicago/Turabian Style

Liu, Jianting, Haifeng Zhong, Chengjuan Cao, Yuqian Wang, Qianrong Zhang, Qingfang Wen, Haisheng Zhu, and Zuliang Li. 2024. "Identification of AP2/ERF Transcription Factors and Characterization of AP2/ERF Genes Related to Low-Temperature Stress Response and Fruit Development in Luffa" Agronomy 14, no. 11: 2509. https://doi.org/10.3390/agronomy14112509

APA Style

Liu, J., Zhong, H., Cao, C., Wang, Y., Zhang, Q., Wen, Q., Zhu, H., & Li, Z. (2024). Identification of AP2/ERF Transcription Factors and Characterization of AP2/ERF Genes Related to Low-Temperature Stress Response and Fruit Development in Luffa. Agronomy, 14(11), 2509. https://doi.org/10.3390/agronomy14112509

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