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Article

Genome-Wide Identification of AUX/IAA Genes in Watermelon Reveals a Crucial Role for ClIAA16 during Fruit Ripening

by
Qi Hu
1,2,
Jingjing Yang
1,
Linghua Meng
1,
Junwei Liu
1 and
Shouwei Tian
1,*
1
State Key Laboratory of Vegetable Biobreeding, Beijing Key Laboratory of Vegetable Germplasms Improvement, Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
2
College of Horticulture, Shanxi Agricultural University, Jinzhong 030810, China
*
Author to whom correspondence should be addressed.
Horticulturae 2023, 9(11), 1167; https://doi.org/10.3390/horticulturae9111167
Submission received: 16 September 2023 / Revised: 19 October 2023 / Accepted: 24 October 2023 / Published: 26 October 2023
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
Browse Figures
Versions Notes

Abstract

:
The auxin/indole-3-acetic acid (Aux/IAA) gene family plays a critical role in auxin-mediated responses and fruit development. However, studies on its role in watermelon are limited. In this study, 29 ClIAA gene members were identified in the watermelon genome and classified into eleven groups. Of note, ClIAA16, which was found to be up-regulated during fruit ripening, was targeted using CRISPR/Cas9 gene editing. Knockout mutants of ClIAA16 exhibited a 3–4 day delay in ripening compared to the wild type, highlighting the regulatory importance of ClIAA16. Our findings shed light on the importance of ClIAA genes in watermelon fruit ripening and pave the way for further functional studies.

1. Introduction

Watermelon (Citrullus lanatus L.), a globally significant fruit crop, not only serves as a valuable source of vitamins and minerals but also provides a useful model for investigating fruit development due to its distinctive characteristics [1]. In plants, the intricate process of fruit development is regulated by various hormones, particularly auxin, which govern crucial stages, such as fruit initiation, growth, expansion, and ripening [2,3]. The Aux/IAA gene family, an early auxin-responsive group, holds a central role in auxin-mediated responses [4]. The Aux/IAA gene family has been detected in numerous fruit genomes, such as 25 in tomato (Solanum lycopersicum) [5], 29 in cucumber (Cucumis sativus) [6], 18 in papaya (Carica papaya L.) [7], 42 in apple (Malus domestica) [8], and 19 in Japanese apricot (Prunus mume) [9]. Nevertheless, the available information regarding this gene family in watermelon is limited.
Aux/IAA proteins are short-lived and predominantly nuclear, contain four conserved domains, I, II, III and IV, although several proteins lacking one or more of these domains are also included in the family [4,10]. Domain I, which contains a leucine repeat motif (LxLxL), functions as a repressor domain and is essential for the recruitment of the transcriptional co-repressor TOPLESS [11]. Domain II plays a crucial role in the instability of Aux/IAA proteins by interacting with the TIR1 protein, a component of the SCFTIR1 ubiquitin ligase, thereby promoting Aux/IAA and facilitating the rapid degradation of Aux/IAA proteins [12,13,14]. Domains III and IV located at the C-terminus serve as dimerization areas that participate in both homo- and hetero-dimerization with other Aux/IAAs and auxin response factor (ARF) proteins [15].
Multiple lines of evidence suggest that Aux/IAAs play crucial roles in the process of fruit development and ripening [16]. The SlIAA9 protein serves as a pivotal mediator of auxin during fruit setting. The inhibition of SlIAA9 leads to the development of simple leaves instead of the wild-type compound leaves and triggers fruit development prior to fertilization, resulting in parthenocarpy [17]. Both ovule and pollen experience a significant reduction in fertility in SlIAA27 down-regulated lines, and the flower and fruit’s internal anatomy is altered, resulting in a larger placenta in smaller fruits [18]. The down-regulation of SlIAA17 resulted in larger fruit diameter, greater volume, and heavier weight compared to the wild type. This increase in fruit size was attributed to a thicker pericarp rather than larger locular spaces [19,20]. The levels of FaAux/IAA1 and FaAux/IAA2 transcripts exhibited a significant increase during the initial phase of fruit development, followed by a sharp decline during the ripening stage (post-white stage). This observation suggests that the expressions of both the FaAux/IAA1 and FaAux/IAA2 genes likely play a role in early fruit development. Furthermore, the up-regulation of FaAux/IAAs transcripts may be partially attributed to auxin-induced fruit growth and the delayed ripening process in strawberry [21]. Additionally, similar key Aux/IAA genes associated with fruit ripening have been identified in Japanese apricot and papaya [7,9]. In a recent study, the utilization of BSA-Seq sequencing in conjunction with transcriptome data analysis unveiled the potential role of an IAA protein (cla004102) and an ethylene response factor (cla004120) as putative regulators of watermelon flesh firmness [22]. Similar observations have been reported in peach [23], apple [24], and durian [25] fruits, where the interplay between auxin and ethylene appears to coordinate the regulation of fruit ripening.
Research on Aux/IAA genes in watermelon is currently limited, which hampers a thorough comprehension and utilization of this gene family’s role in regulating watermelon fruit ripening. This study employed genome-wide mining and identification techniques to acquire a total of 29 members of the ClIAA gene family in watermelon. Bioinformatics methods, such as analyzing chromosome locations, gene structure, conserved motifs, cis-acting elements, and expression profiles, were utilized to investigate the potential biological functions of these genes. Through the analysis of transcriptomic data [26] pertaining to fruit development, we have successfully identified a gene, CLIAA16, which exhibits up-regulation during the process of fruit ripening. In order to gain a deeper understanding of the involvement of ClIAA16 in the ripening process of watermelon fruit, we employed CRISPR/Cas9 gene-editing technology to disrupt the ClIAA16 gene and subsequently obtained mutant variants. In comparison to the wild-type watermelon, the Cliaa16 knockout mutants exhibited a significant delay of 3–4 days in the ripening of fruit. This finding highlights the crucial regulatory function of ClIAA16 in the ripening process of watermelon fruit. The outcomes of this study offer valuable insights into the involvement of ClIAA genes in watermelon fruit ripening and establish a basis for future investigations that may contribute to the understanding of gene functionality in watermelon.

2. Materials and Methods

2.1. Identification and Characterization of ClIAA Genes

The Aux/IAA protein sequence information of Arabidopsis and watermelon ‘97103′ were downloaded from TAIR 10 (http://www.arabidopsis.org, (accessed on 14 July 2022)) and CuGenDBv2 [23] (http://cucurbitgenomics.org/v2/, (accessed on 14 July 2022)) websites, respectively. The Hidden Markov Model file (PF02309) was downloaded from the Pfam database (Pfam: Home page (xfam.org)), and the HMM search software v2.41.2 [27] was used to search all protein sequences in the watermelon genome, screening for candidate family genes with e-value < 1 × 10−5. Candidate family genes were selected in the SMART, CDD, and Pfam databases that contain gene family structural domains. Finally, the candidate gene family genes were determined and named ClIAA1 to ClIAA29 according to their position on the chromosomes. The physical and chemical indicators of watermelon ClIAA gene family proteins (protein sequence length, molecular weight, and isoelectric point) were calculated using bioperl [28]. The WolfPSORT [29] database (https://wolfpsort.hgc.jp, (accessed on 31 July 2022)) was used for subcellular localization analysis, resulting in Table 1.

2.2. Identification and Characterization of ClIAA Genes

Based on the Aux/IAA genomic data information from the watermelon database, MapChart v2.32 [30] was used to draw the chromosome localization map of the ClIAA gene family to observe their distribution on the chromosomes.

2.3. Identification and Characterization of ClIAA Genes

Based on the identified gene family gene ID information, the protein sequences were extracted, and multiple sequence alignment was performed using the muscle software [31]. The identified watermelon ClIAA family genes, along with the Arabidopsis and tomato family genes, were used to construct a phylogenetic tree using the NJ method in MEGA 11 software [32].

2.4. Conserved Motifs Analysis of ClIAA Genes

The MEME software v5.5.4 [33] was used to analyze the conserved base sequences shared by the Aux/IAA gene family. Finally, the gene family gene phylogenetic tree and motifs analysis were combined to further display the gene structure and evolutionary relationships among the gene family members.

2.5. Promoter Cis-Element Analysis of ClIAA Genes

The cis-acting elements in the upstream 1500 bp sequence of the gene family members were analyzed using the Plantcare [34] online website, and the figures were created using the GSDS [35] online software.

2.6. Multiple Sequence Alignment and Collinearity Analysis of ClIAA Genes

The genedoc software v2.7 was used for the multi-sequence alignment analysis of proteins. The protein sequences of the watermelon Aux/IAA family genes were submitted to the STRING database (https://string-db.org/, (accessed on 15 August 2022)) [36] to query the interaction relationships between genes, and the protein interaction network diagram was drawn using cytoscape. An interspecies collinearity analysis and diagramming were performed using the python version of mcscan [37]. The default parameters of mcscanX [38] were used to check for gene doubling and duplication, and the results were displayed using the circos software v0.63-10 [39].

2.7. RNA-seq Analysis of ClIAA Genes Expression

Using existing published watermelon RNA-seq data (SRP012849) [40], we obtained the Aux/IAA genes and their expression data in watermelon at different time periods (10, 18, 26, and 34 days post pollination (DAP)) and in different tissues. The pheatmap software v1.0.12 was used to draw a clustered heatmap of the expression amounts of the watermelon Aux/IAA family genes.

2.8. Construction and Transformation of the CRISPR/Cas9 Vector

The transformation vector was conducted using the binary CRISPR/Cas9 vector pBSE401, as described by Xing et al. [41]. In summary, the PCR forward and reverse primers (DT1 and DT2) were designed to incorporate the two target sites. The resulting PCR fragment was purified and ligated into pBSE401, using the pCBC-DT1T2 plasmid as the template. The constructed vector, pBSE401-IAA16, was then confirmed through Sanger sequencing.
The watermelon explants were subjected to transformation using the method described by Tian et al. [42]. Specifically, cotyledons lacking embryos were dissected into fragments and utilized as explants. The transformation process involved the utilization of the Agrobacterium tumefaciens strain EHA105, which carried the binary vector pBSE401-IAA16. Following co-cultivation of the cotyledon explants in darkness for a duration of 4 days, they were subsequently transferred onto a selective induction medium. Subsequently, the plants were transferred to a selective elongation and rooting medium to obtain intact T0 transgenic plants. Cliaa16 homozygous mutants were identified through Sanger sequencing in T1 generation plants for further phenotypic analysis.

3. Results

3.1. Identification of Aux/IAA Family Genes in the Watermelon Genome

In order to identify the members of the Aux/IAA gene family, a Hidden Markov Model (HMM) was employed to conduct a BLASTp search on proteomic sequences of watermelon. Candidate gene families exhibiting an e-value < 0.00001 were chosen for subsequent analysis. Subsequently, duplicate sequences and those lacking any initiation or termination codons were eliminated, resulting in a set of preliminary candidate genes for the Aux/IAA gene family. The final candidates were obtained from the SMART, NCBI-CDD, and Pfam databases based on the presence of the conserved domain associated with the gene family. A total of 29 gene sequences were found to match the specified characteristics (Table 1).
The watermelon Aux/IAA proteins showed a significant variation (discrepancy) in the protein sequence length. The minimum protein sequence length was observed to be 160 aa, and the largest was observed to be 1491 aa. The relative molecular weight ranged from 16179.7 Da (ClIAA20) to 163519.8 Da (ClIAA5). The theoretical isoelectric point was between 4.52 (ClIAA9) and 9.45 (ClIAA6). Table 1 lists the details information of the genes.

3.2. Chromosomal Localization of ClIAA Genes in Watermelon

To express the distribution of gene family members on chromosomes more intuitively, we mapped the locations of the 29 ClIAA gene DNA sequences on chromosomes using the MapChart software v2.32, creating a chromosomal location map for the gene family. The results showed that the member of genes on each chromosome was not evenly distributed. The widest of the ClIAA genes was observed on Chr06 (seven genes), while no ClIAA genes were identified on chromosomes 3, 4, or 10. Moreover, most genes were located at the upper and lower ends of the chromosomes, and only two genes were found in the middle of the Chr06 (Figure 1).

3.3. Phylogenetic Relationships of Aux/IAA Genes in Watermelon, Tomato, and Arabidopsis

To investigate the phylogenetic relationships and biological associations among members of the watermelon ClIAA gene family, a phylogenetic tree was constructed using the MEGA 11 software (Figure 2). This tree included AUX/IAA gene family members from watermelon, tomato, and Arabidopsis. The resulting phylogenetic distribution revealed that AUX/IAA proteins were classified into 11 distinct clades, denoted as A–K [5,43]. The number of IAA genes in watermelon (29) is comparable to that of Arabidopsis (29), with a slight excess of 25 genes in tomato. Notably, watermelon exhibited an expansion of two clades (B and C) in comparison to Arabidopsis and tomato. However, the non-canonical clade H, which lacks the conserved domains II, is absent in watermelon as well as tomato [5], whereas it is present in Arabidopsis, where it comprises three members (AtIAA16, AtIAA30, and AtIAA31).

3.4. Gene Structure and Protein Structure Analysis of ClIAA Family Genes

In order to ascertain the motif within the ClIAA gene family and gain a deeper comprehension of the diversity and similarity among its members, the motif detection software MEME was employed to analyze the motif. This analysis was then visualized in conjunction with the phylogenetic tree of ClIAAs (Figure 3).
A multi-sequence alignment of ClIAA proteins was conducted (Figure 4). The results showed four domains in the watermelon ClIAA gene family members: domain I, II, III, and domain IV. Most ClIAA proteins contain all conserved domains with a typical “LxLxL” motif on domain I. Some proteins miss ‘domain II’, such as ClIAA9 and ClIAA28. Additionally, a nuclear localization signal (NLS) was detected in most ClIAA proteins at the end of domain IV. A conserved sequence of domain III was found to have a βαα motif that functions in the dimerization process of Aux/IAA (Figure 4).

3.5. Cis-Elements in Promoter Sequences of ClIAA Genes in Watermelon

To investigate the presence and distribution of cis-elements within the promoter region of the ClIAA genes, we selected the upstream sequence of each ClIAA gene, spanning over 1500 bp from the translation site. These sequences were then analyzed using the PlantCare online tool to identify potential cis-elements. A comprehensive analysis was conducted on a set of nine unique cis-elements, which are known to be involved in developmental and hormonal responses (Figure 5). Among all ClIAA genes, the ethylene response element (ERE) was found to be the most commonly occurring element and was present in 21 ClIAAs. Additionally, the anaerobic induction responsive element (ARE) was identified in 20 ClIAAs, while the AAGAA element was found in 19 genes. Furthermore, the abscisic acid-responsive element was also observed in 16 ClIAAs.

3.6. Expression Analysis of ClIAA Genes at Different Stages of Fruit Development

In order to gain a deeper understanding of the role played by the 29 watermelon ClIAA genes in fruit development and ripening, we conducted an analysis of the expression patterns of these genes in watermelon fruit flesh and other tissues at various time points post pollination. This analysis was based on transcriptome data from a previously published study (PRJNA532463) [40]. The expression of these genes is presented in a cluster format, as depicted in Figure 6.
Tween ClIAA genes can detected in the flesh of watermelon, distributed across different groups as follows: Group A (ClIAA3, 7, 10, and 26), Group B (ClIAA2, 13, 22, and 24), Group C (ClIAA4, 6, 14, and 27), Group D (ClIAA25), Group E (ClIAA15), Group F (ClIAA21 and 29), Group G (ClIAA12 and 18), and Group J (ClIAA8 and 16) (Details of the groups are shown in Figure 2). During fruit development, a majority of these genes exhibit down-regulation in their expression levels. However, two genes, ClIAA16 and ClIAA18, display an up-regulation in expression as the fruit matures, suggesting their potential involvement in promoting fruit ripening. Notably, ClIAA16 exhibits a distinct expression pattern specifically in the fruit flesh. Consequently, the findings highlight the potential significance of ClIAA16 and ClIAA18 in the ripening process of watermelon fruit.

3.7. Targeted Mutagenesis of ClIAA16 Using CRISPR/Cas9 Leads to a Late-Ripening Phenotype

In order to investigate the role of ClIAA16 in the ripening process of watermelon, we employed the CRISPR/Cas9 system to generate a gene-editing mutant of ClIAA16. The utilization of the CRISPR/Cas9 technique has significantly transformed the field of genome editing and has been extensively applied in various organisms, including watermelon [42]. Through the utilization of CRISPR-P v2.0 (http://cbi.hzau.edu.cn/CRISPR2/), we identified two highly specific target sites within the ClIAA16 gene that exhibited high cleavage efficiency and minimal off-target effects [44]. These two target sites were located within the second exon of the ClIAA16 gene, as depicted in Figure 7A. The construct pBSE401-ClIAA16, which incorporated two single-guide RNAs designed to target specific sites of the ClIAA16 gene, was utilized for the transformation process. Agrobacterium tumefaciens-mediated transformation was employed to introduce this construct into the callus of ZZJM, a cultivated watermelon inbred line selected in our laboratory.
In the T1 generation, we successfully obtained homozygous gene-edited lines and subsequently identified the specific types of mutations present. Specifically, line Cliaa16-1 exhibited a successful 1 bp insertion at both target sites, resulting in the premature termination of protein translation. In line Cliaa16-2, a 1 bp insertion was observed only at target 1, also leading to premature termination (Figure 7B). After a period of 36 days post pollination (DAP), wild watermelon fruits reached peak levels of pigment and sugar content in the flesh, accompanied by various changes, including seed ripening and a reduction in the thickness of the mesocarp (Figure 7C). Analysis of the sugar content measurements shows that Cliaa16 mutant fruits have a sugar content at 36 DAP that is only 80% of that observed in wild-type watermelon. This level of sugar accumulation is comparable to that observed in wild-type watermelon fruit at 32 DAP (Figure 7D). The pigment accumulation, seed maturity, and mesocarp thickness of the Cliaa16 mutant at 36 DAP also show similarities to those of wild-type watermelon at 32 DAP. This suggests that the mutation in ClIAA16 potentially delays the ripening of watermelon fruit (approximately 3–4 days) rather than specifically affecting a particular physiological indicator. Consequently, these findings suggest that ClIAA16 may play a critical regulatory role in the ripening process of watermelon fruit.

4. Discussion

Auxin plays an important role in virtually all aspects of plant development [45,46]. Aux/IAA proteins are crucial early responders to auxin, binding ARF proteins to regulate target gene expression [47]. To identify the mechanism of auxin involved in the fruit development and ripening of watermelon, a comprehensive collection of 29 Aux/IAA genes were identified and characterized, and their expression was analyzed.
In the present study, a total of 29 Aux/IAA genes were identified in watermelon. Among these, 24 genes exhibited amino acid sequences consisting of less than 500 residues, which aligns with the findings reported in previous studies conducted on Arabidopsis [43], tomato [5], cucumber [6], and Japanese apricot [9] crops. Conversely, only five genes (ClIAA4, 5, 8, 17, and 29) displayed unusually elongated sequences, ranging from 714 to 1491 amino acids. Notably, the four crucial structural domains of Aux/IAA proteins were found to be confined within the initial 350 amino acids of these aforementioned genes. Based on this observation, it is postulated that these genes may have undergone functional misannotation or genetic fusion events within the genome of watermelon 97103. In the recently published T2T genome of watermelon [48], it was observed that ClIAA4, 5, and 8 were annotated as distinct genes, providing confirmation for our hypothesis. Additionally, ClIAA17 and ClIAA29 were identified as separate lengthy genes in the T2T genome. However, there remains a possibility that they are still inaccurately annotated in the T2T genome or subject to gene fusion, which necessitates further experimental validation.
The process of fruit ripening in fleshy fruits is an intricate and well-coordinated developmental process that is regulated by the synchronized activity of various plant hormones [49]. Specifically, ethylene and ABA have been established as crucial factors in the ripening of climacteric and non-climacteric fruits, respectively [50,51]. Nevertheless, recent research has increasingly presented evidence supporting the involvement of auxin in the ripening of both climacteric and non-climacteric fruits [52,53,54]. Auxin levels has been associated with the onset of ripening [55,56]. The softening of peach fruit during ripening is contingent upon a sudden augmentation in indole-3-acetic acid (IAA) content, which subsequently triggers the activation of the PpACS1 gene associated with ethylene synthesis and the subsequent release of ethylene. Conversely, the absence of softening in hard peaches can be attributed to the inhibition of IAA synthesis during ripening, impeding the customary release of ethylene [55,57]. Two candidates genes, PpIAA1 and PpERF4, were reported to interact with and activate the ethylene biosynthesis genes, which is involved in fruit softening in peach [23]. Similar findings have been documented in watermelon, wherein the putative regulatory functions of an IAA protein (Cla004102) and an ethylene response factor (Cla004120) have been elucidated in relation to watermelon flesh firmness [22]. Additionally, the interplay between auxin and ethylene signaling in governing fruit ripening has been investigated in papaya, with the essential role of the ethylene-induced auxin response factor CpARF2 in papaya fruit ripening being demonstrated [58]. In our study, it was observed that the ethylene response element (ERE) was the most frequently encountered element among all ClIAA genes, with a presence in 21 ClIAAs (Figure 5). This finding suggests a potential collaboration between ethylene and auxin in the regulation of various developmental processes in watermelon, extending beyond fruit development and ripening, through the modulation of AUX/IAA expression.
Previous research has demonstrated the involvement of AUX/IAA genes in the process of fruit development and ripening across various plant species [17,18,19,20,21,22,45]. For instance, the genes SlIAA9 and SlIAA27, both belonging to clades B, play a role in regulating tomato fruit set and size [17,18]. In watermelon, the homologous gene ClIAA22, which corresponds to SlIAA9, exhibits the highest levels of expression during the early fruit stage, indicating a shared expression pattern and biological function. The Cla004102 gene (ClIAA13 in this study) had high homology with SlIAA27 auxin-responsive protein, which is associated with flesh firmness in watermelon [22]. However, the down-regulation of Sl-IAA27 resulted in the alteration of fruit morphology, with the formation of fruits with a modified shape and reduced size [18]. These findings suggest that homologous IAA genes may have a conserved effect across different plant species.
The results of our study demonstrate a substantial increase in the expression of the ClIAA16 gene during the ripening phase of watermelon fruit. This up-regulation of gene activity, combined with the observed delay in ripening in ClIAA16 knockout mutants, emphasizes the crucial involvement of ClIAA16 in the ripening process. However, it is important to acknowledge that our research has certain limitations, such as the lack of identification of the specific molecular partners and downstream genes that interact with ClIAA16. Subsequent investigations may prioritize the exploration of these molecular networks, thereby providing a more comprehensive elucidation of the mechanistic underpinnings of ClIAA16′s functionality. Furthermore, it is noteworthy that ClIAA18, an additional IAA gene, demonstrates a substantial up-regulation in the advanced phases of watermelon fruit development. Consequently, it is imperative to ascertain whether ClIAA18 also exerts a notable influence on the ripening process of watermelon fruit. The examination of the interaction and potential functional overlap between ClIAA16 and ClIAA18 emerges as a crucial area of inquiry that warrants further investigation.

Author Contributions

Conceptualization, S.T.; formal analysis, Q.H. and J.Y.; methodology, Q.H., L.M. and J.L.; writing—original draft preparation, S.T. and Q.H.; writing—review and editing, S.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Beijing Academy of Agricultural and Forestry Sciences (YXQN202204, KJCX20230221, and QNJJ202230).

Data Availability Statement

Not applicable to this article.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The chromosomal position of each ClIAAs gene was mapped according to the watermelon genome.
Figure 1. The chromosomal position of each ClIAAs gene was mapped according to the watermelon genome.
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Figure 2. Phylogenetic analysis of ClIAA genes among different family members of watermelon, tomato, and Arabidopsis shown in red, yellow, and green dots, respectively. The outside circle of the phylogenetic tree shows different clades of the tree.
Figure 2. Phylogenetic analysis of ClIAA genes among different family members of watermelon, tomato, and Arabidopsis shown in red, yellow, and green dots, respectively. The outside circle of the phylogenetic tree shows different clades of the tree.
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Figure 3. Analysis of phylogeny and conserved motif of ClIAAs. The phylogenetic relationship of ClIAAs was constructed using MEGA 11 conserved motif distribution of ClIAAs. Nine conserved motifs were labeled with different colors using the MEME program. The relative position is uniformly shown based on the Kilobase scale at the bottom of the figure.
Figure 3. Analysis of phylogeny and conserved motif of ClIAAs. The phylogenetic relationship of ClIAAs was constructed using MEGA 11 conserved motif distribution of ClIAAs. Nine conserved motifs were labeled with different colors using the MEME program. The relative position is uniformly shown based on the Kilobase scale at the bottom of the figure.
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Figure 4. Protein domain analysis of ClIAA family members. The alignment of ClIAA obtained with the genedoc program. The multiple alignment of the domains I–IV of the ClIAA are indicated by blue lines. Color shading indicates identical and conserved amino acid residues. The Nuclear Localization Signal (NLS) is indicated by green lines. All other information is denoted by red lines.
Figure 4. Protein domain analysis of ClIAA family members. The alignment of ClIAA obtained with the genedoc program. The multiple alignment of the domains I–IV of the ClIAA are indicated by blue lines. Color shading indicates identical and conserved amino acid residues. The Nuclear Localization Signal (NLS) is indicated by green lines. All other information is denoted by red lines.
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Figure 5. Various cis−elements were identified using the Plantcare database. The different cis−elements biological terms are shown in different colors. AuxRR: Auxin response region; DRE: Dictyostelium Repetitive Element; TGA: auxin−responsive cis−acting element.
Figure 5. Various cis−elements were identified using the Plantcare database. The different cis−elements biological terms are shown in different colors. AuxRR: Auxin response region; DRE: Dictyostelium Repetitive Element; TGA: auxin−responsive cis−acting element.
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Figure 6. Expression profile of ClIAAs in different tissues and different periods of watermelon based on transcriptome data. Color key shows the expression as red (up-regulated) and blue (down-regulated) of the genes. DAP: Days post pollination.
Figure 6. Expression profile of ClIAAs in different tissues and different periods of watermelon based on transcriptome data. Color key shows the expression as red (up-regulated) and blue (down-regulated) of the genes. DAP: Days post pollination.
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Figure 7. Construction, genotyping and phenotyping of the Cliaa16 mutants. (A) A schematic description of the ClIAA16. Target 1 and target 2 are recognized by gRNA1 and gRNA2, respectively. (B) Targeted mutagenesis of ClWIP1. Sequence alignments of two Cliaa16 T1 homologous mutated lines with the wild-type ClIAA16. (C) Phenotypes of the Cliaa16 mutants. Bar = 5 cm. (D) Mean Brix (a measure of sweetness) of the WT and Cliaa16 fruits. Error bars show the SD of three independent replicates. ** p < 0.01, as determined by Student’s t-test. “ns” means “no significant difference”.
Figure 7. Construction, genotyping and phenotyping of the Cliaa16 mutants. (A) A schematic description of the ClIAA16. Target 1 and target 2 are recognized by gRNA1 and gRNA2, respectively. (B) Targeted mutagenesis of ClWIP1. Sequence alignments of two Cliaa16 T1 homologous mutated lines with the wild-type ClIAA16. (C) Phenotypes of the Cliaa16 mutants. Bar = 5 cm. (D) Mean Brix (a measure of sweetness) of the WT and Cliaa16 fruits. Error bars show the SD of three independent replicates. ** p < 0.01, as determined by Student’s t-test. “ns” means “no significant difference”.
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Table 1. Physiological and biochemical properties of ClIAA genes.
Table 1. Physiological and biochemical properties of ClIAA genes.
Gene NameGene IDLength (aa)MW (Da)pIChromosomal LocationSubcellular Localization
ClIAA1Cla97C01G00470026830,9959.43Chr01:4.51 MbNucleus
ClIAA2Cla97C01G01739038041,525.26.72Chr01:30.97 MbNucleus
ClIAA3Cla97C02G04242019922,193.17.8Chr02:30.37 MbNucleus
ClIAA4Cla97C02G04243071477,740.26.12Chr02:30.4 MbCytoplasm
ClIAA5Cla97C05G0845601491163,519.88.17Chr05:3.51 MbNucleus
ClIAA6Cla97C05G08570026729,028.29.45Chr05:4.22 MbChloroplast
ClIAA7Cla97C05G08571019821,677.34.89Chr05:4.24 MbNucleus
ClIAA8Cla97C05G097930997110,828.36.03Chr05:27.27 MbCytoplasm
ClIAA9Cla97C05G10428017119,776.14.52Chr05:32.19 MbCytoplasm
ClIAA10Cla97C06G11614024127,164.89.09Chr06:7.25 MbNucleus
ClIAA11Cla97C06G11617024726,879.67.79Chr06:7.28 MbChloroplast
ClIAA12Cla97C06G11707037840,587.78.91Chr06:8.48 MbChloroplast
ClIAA13Cla97C06G11863037841,0158.55Chr06:12.83 MbNucleus
ClIAA14Cla97C06G11908023725,627.15.51Chr06:14.96 MbNucleus
ClIAA15Cla97C06G11970016523,228.94.86Chr06:16.99 MbNucleus
ClIAA16Cla97C06G12058024227,430.55.7Chr06:22.77 MbNucleus
ClIAA17Cla97C07G1289001118125,275.55.5Chr07:0.6 MbNucleus
ClIAA18Cla97C07G12967031734,378.28.33Chr07:1.39 MbChloroplast
ClIAA19Cla97C07G13977018516,7249.41Chr07:27.56 MbChloroplast
ClIAA20Cla97C07G13978017916,179.78.04Chr07:27.56 MbNucleus
ClIAA21Cla97C07G14017032535,277.57.1Chr07:27.97 MbNucleus
ClIAA22Cla97C08G15501035538,997.25.49Chr08:23.07 MbNucleus
ClIAA23Cla97C08G15679016724,095.28.51Chr08:24.48 MbCytoplasm
ClIAA24Cla97C09G16397039242,304.85.58Chr09:1.64 MbNucleus
ClIAA25Cla97C09G16450018316,523.27.43Chr09:2.02 MbNucleus
ClIAA26Cla97C09G17015019421,607.28.29Chr09:6.46 MbNucleus
ClIAA27Cla97C09G17016027029,724.98.42Chr09:6.5 MbNucleus
ClIAA28Cla97C11G21806016017,689.88.23Chr11:23.74 MbChloroplast
ClIAA29Cla97C11G221040954105,786.76.52Chr11:27.12 MbNucleus
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Hu, Q.; Yang, J.; Meng, L.; Liu, J.; Tian, S. Genome-Wide Identification of AUX/IAA Genes in Watermelon Reveals a Crucial Role for ClIAA16 during Fruit Ripening. Horticulturae 2023, 9, 1167. https://doi.org/10.3390/horticulturae9111167

AMA Style

Hu Q, Yang J, Meng L, Liu J, Tian S. Genome-Wide Identification of AUX/IAA Genes in Watermelon Reveals a Crucial Role for ClIAA16 during Fruit Ripening. Horticulturae. 2023; 9(11):1167. https://doi.org/10.3390/horticulturae9111167

Chicago/Turabian Style

Hu, Qi, Jingjing Yang, Linghua Meng, Junwei Liu, and Shouwei Tian. 2023. "Genome-Wide Identification of AUX/IAA Genes in Watermelon Reveals a Crucial Role for ClIAA16 during Fruit Ripening" Horticulturae 9, no. 11: 1167. https://doi.org/10.3390/horticulturae9111167

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