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Article

Genome-Wide Analysis of the Related to ABI3/VP1 Family Genes in Chrysanthemum seticuspe Reveals Their Response Patterns to Exogenous Ethylene Treatment

by
Hua Cheng
1,†,
Yiman Yang
1,†,
Jiayu Li
1,
Sumei Chen
1,2,
Fadi Chen
1,2 and
Jiafu Jiang
1,2,*
1
State Key Laboratory of Crop Genetics and Germplasm Enhancement and Utilization, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of State Forestry and Grassland Administration on Biology of Ornamental Plants in East China, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
2
Zhongshan Biological Breeding Laboratory, Nanjing 210014, China
*
Author to whom correspondence should be addressed.
†
These authors contribute equally to this work.
Horticulturae 2023, 9(12), 1316; https://doi.org/10.3390/horticulturae9121316
Submission received: 7 November 2023 / Revised: 1 December 2023 / Accepted: 5 December 2023 / Published: 7 December 2023
(This article belongs to the Special Issue Horticultural Crops Genetics and Genomics)
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Abstract

:
The transcription factor family RELATED to ABSCISIC ACID INSENSITIVE3 (ABI3)/VIVIPAROUS1(VP1) (RAV) is a plant-specific group of transcription factors that only contain a conserved B3 DNA binding domain or both their own B3 and APETALA2 (AP2) domains belonging to the B3 superfamily, which is vital for plant growth, development, and stress response. Although genome-wide characterization and analysis of the RAV family genes have been conducted in some species, they have not been systematically reported in chrysanthemums. Here, we found six RAV family genes in the diploid Chrysanthemum seticuspe genome. Based on domain similarity and homology comparison analyses, RAV genes in Chrysanthemum were categorized into two clades: Class-I and Class-II. Conserved motif analysis revealed that all CsRAV proteins contained the B3 repression domain. An analysis of cis-acting elements suggested that CsRAV family genes may play parts in light, hormonal, abiotic stress, growth, and developmental processes. Furthermore, quantitative RT-PCR analysis validated that all six CsRAV genes responded to ethylene treatment, whereas the genes in the Class-I clade responded most significantly to ethylene. In summary, the above results provided a conceptual basis for further investigation into the functions of CsRAV genes in C. seticuspe.

1. Introduction

The B3 superfamily is a widespread transcription factor in the plant genome that plays an important role in the developmental process and stress response [1]. It can be classified as four subfamilies on the basis of structural features and functions: REPRODUCTIVE MERISTEM (REM), LEAFY COTYLEDON2 [LEC2]-ABSCISICACIDINSENSITIVE3[ABI3]-VAL (LAV), AUXIN RESPONSE FACTOR (ARF), and RELATED TO ABSCISIC ACID INSENSITIVE3(ABI3)/VIVIPAROUS1(VP1) (RAV) [2]. Most B3 family genes in the ARF and LAV subfamilies have been widely investigated; however, studies on the RAV subfamily genes are relatively scarce. Previous studies have shown that Arabidopsis contains 13 RAV family genes that are classified into Class-I and Class-II clades [3]. The Class-I clade has a conserved APETALA2 (AP2) and B3 structural domain, namely AtRAV1, AtRAV1-Like, AtRAV2/AtTEM2 (TEMPRANILLO2), AtRAV2-Like/AtTEM1, AtRAV3, and AtRAV3-Like [4,5]; The Class-II clade contains one B3 structural domain alone, including AtNGATHA1(AtNGA1), AtNGA2, AtNGA3, AtNGA4, AtNGA-LIKE1, AtNGA-LIKE2, and AtNGA-LIKE3 [6]. In addition, AtRAV1 and AtRAV2 were the first RAV family members to be identified [7].
RAV family genes play significant parts in the plant developmental process. For example, overexpression of RAV1 in Arabidopsis leads to fewer lateral roots and rosette leaves, whereas reduced expression of this gene leads to early flowering [8]. Overexpression of GmRAV1 in tobacco leads to delayed flowering, reduces root elongation, and accelerates senescence [9]. In Arabidopsis, TEM genes inhibit trichome initiation by downregulating downstream genes of trichome formation pathways [10]. OsRAV11 and OsRAV12 are directly implicated in regulating rice carpel differentiation and seed development [11]. Overexpression of the GmRAV gene delays soybean flowering as well as maturity and dwarfism phenotypes [12]. Heterologous overexpression of MtRAVs in Arabidopsis promotes meristem formation [13]. Transgenic Arabidopsis overexpressing NGATHA genes (AtNGA1–AtNGA4) lead to small and narrow leaves and flowers, whereas the quadruple mutant nga1/nga2/nga3/nga4 exhibits opposite phenotypes [14]. Suppressor of da1-1 (SOD7)/NGAL2 and DEVELOPMENT-RELATED PcG TARGET IN THE APEX4 (DPA4/NGAL3) negatively regulated seed size by directly inhibiting the expression of KLUH (KLU) [15].
The RAV family genes participate in the abiotic stress response as well, including drought, salt, cold, and so on. Previous studies have shown that downregulation of NtRAV-4 increases drought tolerance in tobacco [16]. Heterologous overexpression of GmRAV-03 in Arabidopsis improves resistance to high salinity and drought [17]. In cucumber, overexpression of CsRAV1 promotes plant tolerance to salt [18]. In cassava, MeRAV5 enhances drought tolerance by regulating hydrogen peroxide and lignin accumulation [19]. The expression level of BnaRAV-1-HY15 is induced by cold, NaCl, and PEG treatments, suggesting that it likely plays a significant role in the response to cold, salt, and drought [20].
In addition, RAV genes are involved in various hormone responses, such as brassinosteroid and ethylene [8,21]. Ethylene is the first phytohormone to be discovered, which is associated with diverse developmental processes and abiotic stress responses [22]. For example, ethylene participates in seed germination, root development, and shoot growth [23]. It also breaks seed dormancy [24], inhibits stamen development [25] and anthocyanin synthesis [26], promotes or inhibits flowering [27,28], and mediates the senescence of leaves and petals [29,30,31]. In grapes, ethylene levels significantly increase under low-temperature stress, which indicates that it participates in modulating cold tolerance [32]. Ethylene likewise increases the heat tolerance of cotton [33]. In addition to its response to temperature stress, ethylene is involved in processes such as flooding [34], drought [35], and salt stress [36]. In addition, a few studies have reported that some RAV subfamily members are involved in ethylene-responsive processes and thus regulate plant growth and development. For instance, the RAV family genes ETHYLENE RESPONSE DNA BINDING FACTOR1 to 4 (EDF1–EDF4) in Arabidopsis are involved in ethylene-mediated petal abscission [30], and the AtEDF1/2/4 genes, which act downstream of the embryonic leaf identity gene FUSCA3, participate in ethylene-mediated developmental transitions [37]. In addition, recent findings have revealed that the chrysanthemum RAV family transcription factor CmTEM1 takes part in the ethylene-mediated floral repression by targeting the APETALA1/FRUITFULL-like1 (CmAFL1) gene directly [38]. Although all of the above studies revealed that RAV genes are involved in ethylene-mediated growth and developmental processes, whether they are involved in ethylene-mediated stress responses needs to be followed up.
Chrysanthemum (Chrysanthemum morifolium Ramat.) has significant market value and cultural heritage. At present, the majority of chrysanthemums supplied on the market are hexaploid cultivated chrysanthemums; their genomes are highly heterozygous and their genetic backgrounds are complex [39], which present a considerable research challenge. The diploid chrysanthemum (Chrysanthemum seticuspe) is the model species of hexaploid chrysanthemum, which is homologous to hexaploid chrysanthemum, and the establishment of the genetic transformation system of C. seticuspe has recently been accomplished by researchers [40]. Therefore, in this study, we selected the genome of this species for conducting follow-up studies. So far, a genome-wide analysis of the RAV gene family has been accomplished in soybean [17], rice [41], and wheat [42]; however, it has not yet been analyzed in chrysanthemums, particularly in terms of ethylene response analysis at a genome-wide level. Here, we performed a genome-wide analysis of the RAV genes in C. seticuspe and explored their response patterns to ethylene treatment, which provides candidate genes for using ethylene to regulate the growth and development of chrysanthemum and enhance the ability of environmental adaptation in the future.

2. Materials and Methods

2.1. Plant Materials and Ethephon Treatment

Seedlings of Gojo-0 were planted in a mixed substrate of nutrient soil: vermiculite (3:1) and cultured in a growth chamber under long-day conditions (16 h light and 8 h dark, 22 °C, 70 μmol photons/m2/s). Plants grown to the 12–13 fully expanded leaf stage were used for ethephon (an ethylene release agent) and water treatments. The steps were as follows: First, the whole plant was sprayed with water (as the control) or 100 mg/L ethephon. Subsequently, the treated plants were sealed in 14 L transparent plastic boxes. Samples were collected at 0, 1, 3, 6, and 12 h. Three independent monocultures were mixed for use as one biological replicate, and the experiment consisted of three independent biological replicates.

2.2. Total RNA Isolation and qRT-PCR Analysis

The methods of total RNA isolation and experimental procedures of qRT-PCR were similar to Guan et al. [43]. The expression levels were normalized against CsEF1α loading standards using the 2−ΔΔCT method [44]. Three biological replicates were performed. The primers employed for this research were designed using the Primer Designing Tool website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 22 October 2023) and were presented in Table S1.

2.3. Identification and Physicochemical Property Analysis of CsRAV Family Genes in C. seticuspe

C. seticuspe genome sequences and annotation data were accessible to the PlantGarden website (https://plantgarden.jp/en/list/t1111766/genome; accessed on 2 October 2023). The protein sequences of the RAV family in Arabidopsis were obtained from the Tair website (https://www.arabidopsis.org/; accessed on 2 October 2023).
Three steps were performed to identify the C. seticuspe RAV family genes. First, we downloaded the hidden Markov models (HMMs) of AP2 (PF00847) and B3 (PF02362) from the Pfam database (https://pfam.xfam.org/; accessed on 3 October 2023) and employed the above queries to search against C. seticuspe genome using HMMER 3.3.1 software with an E-value < 1 × 10−5 [42]. Second, we used all A. thaliana RAV proteins to blast against the sequences obtained in the first step with E < 1 × 10−5 to obtain the putative CsRAV genes. Third, we used the Simple Modular Architecture Research Tool (SMART) online website (http://smart.embl-heidelberg.de/; accessed on 3 October 2023) to further confirm the putative CsRAVs, and sequences without filtering out the AP2 and/or B3 domains. Subsequently, the C. seticuspe RAV family genes were obtained.
In addition, we calculated various protein properties, including the number of amino acids, theoretical isoelectric point (pI), molecular weight, instability index, grand average hydrophilicity (GRAVY), and subcellular localization. Calculations were performed by the ExPASY website (http://web.expasy.org/protparam/; accessed on 3 October 2023). Next, we predicted the subcellular localization using Cell-PLoc-2 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) and Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/), both accessed on 3 October 2023.

2.4. Phylogenetic Tree Construction and Multiple Sequence Alignment

For constructing the phylogenetic tree, the protein sequences of the RAV family from A. thaliana and C. seticuspe were previously obtained [4,6,45]. The MEGA 11 software [46] was used to construct the phylogenetic tree with the neighbor-joining (NJ) method employing the bootstrap method with 1000 replicates for phylogeny testing. Subsequently, a phylogenetic tree was visualized using the R package ggtree. Multiple sequence alignment using ClustalX 2.1 [47].

2.5. Protein and Gene Structure Prediction of CsRAVs

To predict protein secondary structures, we utilized the SOPMA online software (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html; accessed on 4 October 2023). We performed these predictions for specific CsRAV proteins using unique job IDs.
To predict the tertiary structure of protein conserved domains, we employed the SWISS-MODEL website (https://swissmodel.expasy.org/; accessed on 4 October 2023), creating models for the CsRAV proteins. We also used the SMART website for the structural domain identification of CsRAV proteins, and the link to access the website has been provided above.
To analyze the conserved motifs in CsRAV proteins, we used the Multiple Em for MEME Suite (MEME) 5.5.4 online program (https://meme-suite.org/meme/doc/meme.html; accessed on 5 October 2023). Exon–intron positions and conserved motifs were visualized using Tbtools [48].

2.6. Promoter Sequence Analysis and Tissue Expression Analysis of CsRAV Family Genes

We obtained the 2 kb sequences upstream from the ATG start codon of the CsRAV genes from the C. seticuspe genome. Cis-acting elements analyses in the CsRAVs promoters were performed by the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/; accessed on 6 October 2023). The final analysis outcomes were visualized and displayed by employing TBtools. We utilized the R package ggplot2 to generate a statistical visualization of the elements within CsRAVs promoters.
The expression patterns of the CsRAV gene family among different tissues were obtained from PRJNA82048835 [49], and TBtool software was used to create a heat map.

2.7. Statistical Analysis of Data

The significance analysis of the data was performed using SPSS 25 (IBM, New York, NY, USA) with Student’s t-test (* p < 0.05). Error bars showed standard deviation (SD).

3. Results

3.1. Identification and Physicochemical Properties of CsRAV Genes in C. seticuspe

We identified six CsRAV genes by searching the C. seticuspe genome in total, namely CsNGA2, CsNGA-Like2, CsNGA1, CsNGA-Like1, CsTEM1, and CsTEM2 (Table 1). In addition, in order to further understand the CsRAV family genes, we investigated their physicochemical properties, and the number of amino acids, theoretical pI, molecular weight of protein, instability index, grand average of hydrophilicity (GRAVY), and subcellular localization were also analyzed (Table 1). The results revealed that the amino acid length ranged from 225 aa (CsNGA-Like1) to 465 aa (CsNGA1), with a mean length of 348 aa. The molecular weights of these proteins ranged from 25.6 to 51.70 kDa, and the average protein molecular weight was 39.16 kDa. The theoretical pI values ranged from 6.28 to 9.52, with the highest for CsTEM2 (9.52) and the lowest for CsNGA1 (6.28). The pIs of CsNGA-Like1, CsNGA1, and CsNGA-Like2 were <7, indicating that these proteins were acidic, whereas those of CsTEM1, CsTEM2, and CsNGA2 were >7, indicating that these proteins were basic. The GRAVY values of the CsRAV proteins were negative, indicating that all CsRAVs were hydrophilic proteins. Protein instability index analysis suggested that except for CsTEM1 (39.72), which was a stable protein, the other five proteins were unstable. Subcellular localization prediction indicated that all members of this family were localized in the nucleus, implying that they mainly perform their functions in the nucleus.

3.2. Phylogenetic Tree and Multiple Sequence Alignment Analyses of CsRAV Family Genes

To explore the evolutionary relationships of the RAV family genes between A. thaliana and C. seticuspe, a phylogenetic tree of six CsRAVs and thirteen AtRAVs was constructed using the neighbor-joining (NJ) method. The results demonstrated that CsTEM1 and CsTEM2 had a higher homology compared to AtTEM1 and AtTEM2, which clustered into the Class-I clade, whereas the other four members of the CsRAV family genes were classified into the Class-II clade (Figure 1).
Furthermore, we conducted a multiple sequence alignment analysis of RAV family genes in A. thaliana and C. seticuspe. The results indicated that all the Class-I proteins contained a conserved AP2 and B3 domain (Figure 2a), and the proteins classified as the Class-II clade had only one B3 domain (Figure 2b). Interestingly, we found that except for AtRAV3 and AtRAV3-like proteins, all other proteins contained the typical B3 repression domain (BRD) in the C-terminal region (Figure 2). In summary, these results revealed that all CsRAV family proteins contain BRD motifs, suggesting that they may act as transcriptional suppressors.

3.3. Predictions of Secondary and Tertiary Structural Characteristics of CsRAV Proteins

Studying protein structures contributes to understanding the function of proteins, and interactions between proteins (or other molecules). Therefore, we analyzed the secondary and tertiary structures of CsRAV proteins. The results showed that the secondary structure of CsRAV proteins includes α-helix, β-folding, extended chains, and random crimp (Table S2; Figure S1). The proportion of random crimp was the highest (>50%); β-folding accounted for the lowest proportion, ranging from 3.87 to 7.52%; α-spiral accounted for 5.33–24.12%; and extended chain accounted for 18.53–21.78%. The outcomes of the tertiary structure prediction suggested that the tertiary structures of four proteins, excluding CsTEM1 and CsTEM2, were different, indicating that the biological functions of CsTEM1 and CsTEM2 may be similar (Figure 3).

3.4. Analyses of Gene Structure of CsRAV Genes and Its Encoding Protein Conserved Motifs

In the evolutionary process of multigene families, the multifariousness of the gene structure is an inevitable condition responsible for the evolution of genes to acquire novel functions to adapt to changing environments [50,51]. To understand the structural variety of CsRAV genes, we dissected the intron/exon structure of the genes in this family. The results indicated that among the six CsRAV genes, CsTEM1 and CsNGA2 had no introns, CsTEM2 and CsNGA1 had only one intron, and CsNGA-Like1 and CsNGA-Like2 had the same gene structure, both containing two introns (Figure 4a).
Moreover, we also used the MEME motif search tool to analyze the conserved motifs of CsRAV proteins (Figure 4b,c). The results suggested that the distribution of motifs belonging to the same subfamily was consistent. In addition, we found that motifs 1, 2, 4, and 8 were present in all CsRAV proteins, motifs 1 and 2 constituted the B3 domain, and motif 4 was a typical BRD inhibitory motif. Motifs 3 and 6 are a part of the AP2 domain. In addition, motif 5 was found only in the Class-II clade, and motif 7 was found only in the Class-I clade, suggesting that specific motifs may determine their own unique functions.

3.5. Analysis of Cis-Acting Elements in CsRAV Gene Family

Cis-acting elements are binding sites for the transcription factors involved in plant growth, differentiation, and development. Here, we conducted a cis-acting elements analysis on the 2.0 kb promoter region upstream of the CsRAV genes’ initiation codon using the PlantCARE database (Figure 5). The outcomes indicated that apart from the general transcriptional regulatory elements TATA-box and CAAT-box, as well as unknown functional elements, an amount of 161 cis-acting elements were discovered. Subsequently, we evaluated the detailed statistics of these elements. There were 71 light response elements (44.10%), including Box4, I-box, G-box, and so on. Further, 51 (31.68%) hormone response elements were identified, including ethylene response elements (ERE), abscisic acid response elements (ABRE), and methyl jasmonate response elements (TGACG-motif and CGTCA-motif). A total of 25 abiotic stress response elements (15.6%), including the drought-responsive element (DRE), low-temperature response elements (LTR), anaerobic inducible elements (ARE), and the WUN-motif, were followed by hormone response elements. In addition, only 14 elements (8.70%) were related to growth and development responses. In brief, CsRAVs may play a significant role in phytohormone and light responses in plants and may also be implicated in abiotic stress responses and developmental processes.

3.6. Expression Profiling of CsRAV Genes across Different Tissues

The function of a gene is often closely associated with its expression pattern [52]. In our study, the RNA sequencing data published by Sun et al. [49] were used to analyze the tissue-specific expression profiles of CsRAV genes. The results, shown in Figure 6 and Table S3, suggested that CsTEM1 is constitutively expressed in all organs. CsTEM2 and CsNGA-Like1 were only expressed in the tissues of vegetative roots, stems, and leaves but not in ray and disc flowers during the reproductive growth stage (FPKM > 0.3 was considered as the expressed gene). However, CsNGA2 was only expressed in the ray and disc flowers and was not expressed in any tissue at the vegetative growth stage. In addition, we found that two genes, CsNGA-Like2 and CsNGA1, were not expressed in the stem but were highly expressed in other tissues. Interestingly, we discovered that all CsRAV genes except CsNGA2 were highly expressed in the roots. These results led us to speculate that CsRAV genes may participate in the regulation of diverse physiological and biochemical processes, including root growth and development.

3.7. Expression Analysis of CsRAVs to Ethylene Treatment

Previous studies have shown that four AtRAV genes (AtRAV2, AtRAV2-like, AtRAV1, and AtRAV1-like) are involved in ethylene signaling pathways and are upregulated by ethylene induction [30,37]. In this study, the response of CsRAV genes to ethylene treatment was investigated using qRT-PCR. The conclusions suggested that the response of CsRAV genes was different to ethylene treatment. Two genes, CsTEM1 and CsTEM2, belonging to the Class-I clade were upregulated by ethylene induction, especially at 12 h after treatment (Figure 7a,b). In the Class-II clade, CsNGA1 and CsNGA2 were inhibited by ethylene in the early stages of treatment and did not respond to ethylene at 12 h after treatment (Figure 7c,d), CsNGA-Like1 and CsNGA-Like2 were upregulated at 3 h and 12 h of ethylene treatment, respectively (Figure 7e,f). The results showed that all CsRAV genes were significantly involved in ethylene response, and the response trends of the two genes with high homology were consistent.

4. Discussion

The RAV family of genes is a plant-specific family of transcription factors that play vital roles in growth and development, hormones, and stress responses, as reported in soybean [17], Arabidopsis [13], rice [41], and wheat [42]. However, relatively little research has been conducted on RAV family genes in chrysanthemums. In the current study, the genome-wide analysis revealed six RAV family genes in C. seticuspe (Table 1), in comparison with the eight reported in cucumber [18], thirteen in Arabidopsis [2], and twenty-six in wheat [42], indicating that the number of RAV family genes differs greatly among different species, and this difference in number may be due to their adaptation to different biological functions or environmental conditions. Moreover, we found that two of the six genes in C. seticuspe belonged to the Class-I clade, which contained the AP2 and B3 domains (Figure 1 and Figure 2a), whereas the other four genes (CsNGA1, CsNGA2, CsNGA-Like1, and CsNGA-Like2) only had one B3 domain and belonged to the Class-II clade (Figure 1 and Figure 2b). More interestingly, we found that all of the CsRAV genes contain the canonical B3 repression domain ‘RLFGV’ (Figure 2) [53], indicating that they may play the role of transcriptional repressors. In addition, it has been shown that intron is involved in gene expression and evolution [54]. It has been reported that only 20% of the RAV genes in rice contain introns [41], whereas our results showed that 33.3% of the RAV genes in chrysanthemum were free of introns (Figure 4a), suggesting that introns were gained or lost to different extents in different species during the evolutionary process. However, whether the loss or retention of introns will affect the expression of RAV genes needs to be further investigated.
The cis-acting elements of gene promoters play crucial roles in regulating plant growth, development, and environmental interactions [55]. Therefore, the analysis of cis-acting elements can reveal the transcriptional regulation modes of the corresponding genes and explore their biological functions. Here, we also investigated the cis-acting elements of CsRAV family genes. Surprisingly, the promoters of CsRAVs had the most light-responsive elements (such as G-box, I-box, Box4), accounting for 44.10% (Figure 5), nearly half of which were cis-acting elements, indicating that genes in this family are largely regulated by light signal. It has been shown that RAV genes are associated with abiotic stresses responses, such as MeRAV5, which was involved in regulating hydrogen peroxide and lignin accumulation, thus playing a crucial role in enhancing drought resistance in cassava [19]. AtTEM2, a member of the RAV family genes of Arabidopsis, is downstream of SHORT VEGETATIVE PHASE (SVP) and is involved in the low-temperature pathway regulation of flowering [56]. Our analysis also found some drought and low-temperature response elements on the promoters of CsRAV family genes, indicating that CsRAVs are also involved in the response to drought and low temperatures (Figure 5). So far, an increasing number of studies have reported that RAV family genes are vital for growth and development. Heterologous overexpression of GmRAV1 in tobacco delays its development and flowering [9,57]. In Arabidopsis, RAV1 affects blooming time, leaf senescence, and seed development [8,29,58]. We also found fourteen cis-acting elements connected with growth and development in the promoters of CsRAV genes, indicating that RAV family genes of different species are probably conserved in regulating growth and development. Hormones are important endogenous signals in plants and take part in various developmental processes and environmental stimuli. We also found some hormone response elements in the promoters of CsRAV genes, such as abscisic acid (ABA), Methyl jasmonate (MeJA), salicylic acid (SA), Auxin, and so on (Figure 5). In soybeans, all RAV genes were significantly induced by ABA at the transcriptional level [17]. In cotton, the expression of GhRAV4, GhRAV9, and GhRAV20 was notably induced by Brassinolide (BL), JA, and indole-3-acetic acid (IAA), respectively [59]. Previous research has also revealed that AtTEM1 can directly bind to the promoters of the gibberellin (GA) biosynthetic genes GA3-oxidase1 and 2 (GA3OX1 and GA3OX2) to reduce GA content [60]. Our findings show that CsRAV genes in chrysanthemum may be directly induced by GA.
Apart from the above hormone response elements, we found several ethylene response elements in the promoters of the CsRAV genes as well (Figure 5). Ethylene is a phytohormone that plays an essential role in the plant life cycle, from growth and development to the stress response [61]. Members of the ethylene signaling pathway in Arabidopsis and rice have been identified [61]. In Arabidopsis, four members of the RAV family, the Class-I clade (EDF1-4), are downstream of the ethylene signaling transcription factor ETHYLENE-INSENSITIVE 3 (EIN3) [21,37]. Studies have reported that the EDF4/RAV1 of Arabidopsis positively regulates leaf senescence [29]. Further studies have indicated that EDFs take part in ethylene signaling and regulate Arabidopsis floral organ senescence and abscission [30]. In cassava, anti-ethylene and pruning treatments downregulated TEM1 [62]. Recent studies have shown that ethylene upregulates CmTEM1 in chrysanthemum [38]. In our study, we found that all six CsRAV genes were regulated by ethylene (Figure 7), which accords with RAV genes in other species. However, their interaction with ethylene signaling to regulate plant growth, development, and stress stimuli requires further investigation.

5. Conclusions

In conclusion, this study systematically characterized and analyzed the RAV family genes in C. seticuspe. In total, six CsRAV genes were identified, which contain an AP2 domain and/or a B3 domain, and all of them own a typical BRD motif. Cis-acting element analysis showed that CsRAV genes might be involved in the abiotic stress response, hormone response, and growth and developmental processes. A tissue quantification analysis demonstrated that most of the CsRAV genes were highly expressed in roots. In addition, analysis of ethylene-responsive expression patterns indicated that all genes in this family responded to ethylene. This research establishes a foundation for further exploring the functions of CsRAV genes in C. seticuspe.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae9121316/s1, Table S1. The primer sequences employed in the current study. Table S2. The data of secondary structure of CsRAV protein. Table S3. Raw data of tissue quantitative expression profile of C. seticuspe. Figure S1. The picture of secondary structure prediction of CsRAV protein. Blue, red, green, and yellow lines represent α-helix, extended strands, β-turn, and random coil, respectively.

Author Contributions

Conceptualization, J.J. and H.C.; Investigation, H.C.; Supervision, J.J.; validation, J.L. and Y.Y.; Writing—original draft, H.C. and Y.Y.; Writing—review and editing, S.C., F.C. and J.J. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by grants from the National Natural Science Foundation of China (31930100), the Fundamental Research Funds for the Central Universities (QTPY2023002, KYT2023006), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Data Availability Statement

All relevant data can be found in the manuscript and the supporting materials.

Acknowledgments

We would like to thank Makoto Kusaba, Hiroshima University, for sharing the material of Chrysanthemum seticuspe Gojo-0.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic analysis between C. seticuspe and A. thaliana. Amino acid sequences of RELATED to ABSCISIC ACID INSENSITIVE3 (ABI3)/VIVIPAROUS1(VP1) (RAV) family genes were used to build the tree using MEGA 11 combined with the neighbor-joining (NJ) method. The pink represents Class-I clade and the yellow indicates Class-II clade. Note: gene name initial of ‘At’ are genes of A. thaliana, ‘Cs’ are genes of C. seticuspe. Different shapes represent different species.
Figure 1. Phylogenetic analysis between C. seticuspe and A. thaliana. Amino acid sequences of RELATED to ABSCISIC ACID INSENSITIVE3 (ABI3)/VIVIPAROUS1(VP1) (RAV) family genes were used to build the tree using MEGA 11 combined with the neighbor-joining (NJ) method. The pink represents Class-I clade and the yellow indicates Class-II clade. Note: gene name initial of ‘At’ are genes of A. thaliana, ‘Cs’ are genes of C. seticuspe. Different shapes represent different species.
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Figure 2. Multiple sequence alignment of RAV family genes from C. seticuspe and A. thaliana. (a,b) Multiple sequence alignment of Class-I (a) and Class-II clades (b). The orange and green boxes represent the AP2 and B3 conserved domains, respectively; the blue box represents B3 repression domain (BRD).
Figure 2. Multiple sequence alignment of RAV family genes from C. seticuspe and A. thaliana. (a,b) Multiple sequence alignment of Class-I (a) and Class-II clades (b). The orange and green boxes represent the AP2 and B3 conserved domains, respectively; the blue box represents B3 repression domain (BRD).
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Figure 3. Tertiary structure prediction of CsRAV proteins. (a) CsTEM1, (b) CsTEM2, (c) CsNGA1, (d) CsNGA2, (e) CsNGA-Like1, (f) CsNGA-Like2. The tertiary structures were acquired using SWISS-MODEL online tool (https://swissmodel.expasy.org/, accessed on 4 October 2023).
Figure 3. Tertiary structure prediction of CsRAV proteins. (a) CsTEM1, (b) CsTEM2, (c) CsNGA1, (d) CsNGA2, (e) CsNGA-Like1, (f) CsNGA-Like2. The tertiary structures were acquired using SWISS-MODEL online tool (https://swissmodel.expasy.org/, accessed on 4 October 2023).
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Figure 4. Gene and motif structure analysis of CsRAV family genes were analyzed using Tbtools and MEME. (a) The analysis of CsRAV genes’ structure, including intron and exon distribution. (b) Schematic plots of motif structure of the CsRAV family genes using MEME. Different colors represent eight motifs, (c) Details of the eight motifs of CsRAV family genes.
Figure 4. Gene and motif structure analysis of CsRAV family genes were analyzed using Tbtools and MEME. (a) The analysis of CsRAV genes’ structure, including intron and exon distribution. (b) Schematic plots of motif structure of the CsRAV family genes using MEME. Different colors represent eight motifs, (c) Details of the eight motifs of CsRAV family genes.
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Figure 5. Putative cis-acting elements identified in the promoter region of RAV genes from C. seticuspe. (a,b) Quantity statistics (a) and locations (b) of cis-acting elements on the promoter region of CsRAV genes.
Figure 5. Putative cis-acting elements identified in the promoter region of RAV genes from C. seticuspe. (a,b) Quantity statistics (a) and locations (b) of cis-acting elements on the promoter region of CsRAV genes.
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Figure 6. Heatmap showing expression profiling of CsRAV genes across various tissues. The graph displays the normalized log2 (FPKM + 1) values for each tissue.
Figure 6. Heatmap showing expression profiling of CsRAV genes across various tissues. The graph displays the normalized log2 (FPKM + 1) values for each tissue.
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Figure 7. Expression patterns of CsRAV family genes after water and ethephon treatments. (a–f) The relative expression levels of CsTEM1 (a), CsTEM2 (b), CsNGA1 (c), CsNGA2 (d), CsNGA-Like1 (e) and CsNGA-Like2 (f) following water and ethephon treatments. Control represents water treatment; ethylene indicates 100 mg/L ethephon treatment. Error bars represent SD, Student’s t-test (* p < 0.05).
Figure 7. Expression patterns of CsRAV family genes after water and ethephon treatments. (a–f) The relative expression levels of CsTEM1 (a), CsTEM2 (b), CsNGA1 (c), CsNGA2 (d), CsNGA-Like1 (e) and CsNGA-Like2 (f) following water and ethephon treatments. Control represents water treatment; ethylene indicates 100 mg/L ethephon treatment. Error bars represent SD, Student’s t-test (* p < 0.05).
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Table 1. Characteristic analysis of the RAV proteins in C. seticuspe.
Table 1. Characteristic analysis of the RAV proteins in C. seticuspe.
Cse_IDCse_NameNumber of Amino Acids Theoretical pIMolecular Weight Instability IndexGrand Average of Hydropathicity Predicted Location
CsG_LG1.g11139.1CsNGA2400 aa8.8145.22 kDa67.19−0.755Nucleus
CsG_LG2.g22046.1CsNGA-Like2306 aa6.3435.13 kDa54.52−0.992Nucleus
CsG_LG3.g53814.1CsNGA1465 aa6.2851.70 kDa59.07−0.898Nucleus
CsG_LG8.g07956.1CsNGA-Like1225 aa6.6325.60 kDa49.42−0.669Nucleus
CsG_LG8.g32031.1CsTEM1340 aa9.1237.98 kDa39.72−0.555Nucleus
CsG_LG9.g58287.i1CsTEM2350 aa9.5239.31 kDa52.27−0.667Nucleus
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Cheng, H.; Yang, Y.; Li, J.; Chen, S.; Chen, F.; Jiang, J. Genome-Wide Analysis of the Related to ABI3/VP1 Family Genes in Chrysanthemum seticuspe Reveals Their Response Patterns to Exogenous Ethylene Treatment. Horticulturae 2023, 9, 1316. https://doi.org/10.3390/horticulturae9121316

AMA Style

Cheng H, Yang Y, Li J, Chen S, Chen F, Jiang J. Genome-Wide Analysis of the Related to ABI3/VP1 Family Genes in Chrysanthemum seticuspe Reveals Their Response Patterns to Exogenous Ethylene Treatment. Horticulturae. 2023; 9(12):1316. https://doi.org/10.3390/horticulturae9121316

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Cheng, Hua, Yiman Yang, Jiayu Li, Sumei Chen, Fadi Chen, and Jiafu Jiang. 2023. "Genome-Wide Analysis of the Related to ABI3/VP1 Family Genes in Chrysanthemum seticuspe Reveals Their Response Patterns to Exogenous Ethylene Treatment" Horticulturae 9, no. 12: 1316. https://doi.org/10.3390/horticulturae9121316

APA Style

Cheng, H., Yang, Y., Li, J., Chen, S., Chen, F., & Jiang, J. (2023). Genome-Wide Analysis of the Related to ABI3/VP1 Family Genes in Chrysanthemum seticuspe Reveals Their Response Patterns to Exogenous Ethylene Treatment. Horticulturae, 9(12), 1316. https://doi.org/10.3390/horticulturae9121316

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