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Article

Identification and Expression Analysis of the SKP1-Like Gene Family under Phytohormone and Abiotic Stresses in Apple (Malus domestica)

by
Miao Shao
,
Ping Wang
,
Huimin Gou
,
Zonghuan Ma
,
Baihong Chen
and
Juan Mao
*
College of Horticulture, Gansu Agricultural University, Lanzhou 730070, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(22), 16414; https://doi.org/10.3390/ijms242216414
Submission received: 16 October 2023 / Revised: 12 November 2023 / Accepted: 14 November 2023 / Published: 16 November 2023
(This article belongs to the Section Molecular Plant Sciences)
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Abstract

:
Ubiquitination participates in plant hormone signaling and stress response to adversity. SKP1-Like, a core component of the SCF (Skp1-Cullin-F-box) complex, is the final step in catalyzing the ubiquitin-mediated protein degradation pathway. However, the SKP1-Like gene family has not been well characterized in response to apple abiotic stresses and hormonal treatments. This study revealed that 17 MdSKP1-Like gene family members with the conserved domain of SKP1 were identified in apples and were unevenly distributed on eight chromosomes. The MdSKP1-Like genes located on chromosomes 1, 10, and 15 were highly homologous. The MdSKP1-like genes were divided into three subfamilies according to the evolutionary affinities of monocotyledons and dicotyledons. MdSKP1-like members of the same group or subfamily show some similarity in gene structure and conserved motifs. The predicted results of protein interactions showed that members of the MdSKP1-like family have strong interactions with members of the F-Box family of proteins. A selection pressure analysis showed that MdSKP1-Like genes were in purifying selection. A chip data analysis showed that MdSKP1-like14 and MdSKP1-like15 were higher in flowers, whereas MdSKP1-like3 was higher in fruits. The upstream cis-elements of MdSKP1-Like genes contained a variety of elements related to light regulation, drought, low temperature, and many hormone response elements, etc. Meanwhile, qRT-PCR also confirmed that the MdSKP1-Like gene is indeed involved in the response of the apple to hormonal and abiotic stress treatments. This research provides evidence for regulating MdSKP1-Like gene expression in response to hormonal and abiotic stresses to improve apple stress resistance.

1. Introduction

Ubiquitin (Ub) is a small protein consisting of 76 amino acids, that is highly conserved in eukaryotes. Ubiquitination plays a key role in protein function, regulation, and degradation. Plants can thus rely on ubiquitination in the environment to regulate hormone signaling and stress responses [1]. The process of ubiquitination in plants is mainly regulated by a variety of ubiquitinases, such as ubiquitin-activating enzymes, binding enzymes, ligases, and dissociative enzymes (E1, E2, E3, and DUBS) [2,3]. E3 ligases act as a specific substrate selector in the ubiquitin protease cascade reaction. Structurally, E3 ligases can be divided into single-subunit E3 ligases and multi-subunit E3 ligases [4]. SCF (SKP1/Cullin1/F-box) [5] complex is a class of multisubunit RING E3 ligases consisting of the SKP1 protein, the Cullin1 protein, the RBX1 protein, and a variable F-box protein [6]. Previous work found that FBX proteins can interact with ASK proteins in Arabidopsis [7]. The s-phase kinase-related protein 1 (SKP1), a major component of the SCF complex, plays an important role in ubiquitin-mediated plant-protein degradation [4].
ASK1 was involved in protein degradation during flowering in Arabidopsis [8]. ASK1 can also act with other members of the SCF complex to control plant light signals [9] and was related to the plant’s viral defense [10]. Currently, 21,31,19, and 15 SKP1-Like genes were firmly established in Arabidopsis [11], rice [12], tomato [13], and chickpea [14] species, respectively. SKP1-Like genes have also been identified in several species, such as wheat SKP1 genes, soybean SKP1 genes, pepper SKP1 gene, citrus SKP1 gene, and peony SKP1 gene [15,16,17,18,19].
The SCF complex formed by SKP1 was in response to many hormone regulatory pathways, and TIR1 and ASK1 interacted to form the SCFTIR1 complex, which can regulate Aux/IAA proteins in the hormone-signaling pathway of auxin [20], while the SCFSLEEPY1 complex formed by SLEEPY1 and ASK1 was involved in the regulation of the hormone-signaling pathway of gibberellin [21]. In Arabidopsis, COI1 can act as a jasmonate receptor [22] and some ASK can bind to it to form the SCFCOI1 complex, which is involved in the jasmonate hormone-signaling pathway [23]. SKPs could be related to the hormone signaling pathway of ABA and, thus, respond to abiotic stresses [24,25].
Extensive studies have proven that SKP1-Like genes participated in abiotic stresses in plants. For example, SSK genes in wild tomato plants responded to both heat and salt stress [13]. In soybean (Glycine max), the GmSK1 gene overexpression resulted in increased plant tolerance to high salt and drought stresses [18]. GsSKP21, a SKP1-Like family gene, has a conserved SKP structural domain. GsSKP21 gene overexpression enhanced soybeans’ (Glycine soja) tolerance to alkaline stress and also reduced plant sensitivity to ABA [26]. In peonies, the PSK1 gene overexpression also enhanced the drought tolerance of plants and promoted flower formation and early flowering [17]. In chickpeas, CaSKP genes are also involved in drought, salt, and oxidative stresses in plants [14]. These suggest that SKP1-Like genes may play a crucial role in apple response to abiotic stresses and phytohormone signaling pathways. Therefore, genome-wide exploration of the MdSKP1-Like gene family in response to environmental stresses may provide insights into the precise regulation of tolerance to various environmental stresses and responses to exogenous hormones in apple.
Apples are a major source of fresh food and vitamin supplementation for people daily. However, apple growth is threatened by drought, salinity, and osmotic stress, etc. In this study, the apple SKP1-Like gene family was analyzed and identified using bioinformatics methods. The distribution, gene structure, gene homology, conservation patterns, selection pressure, codon preference analysis, predicted-protein interaction pathways, cis-acting elements, and evolutionary relationships of SKP1-Like genes in apple chromosomes were analyzed at the genomic level. The expression levels of SKP1-Like genes in different apple tissues were analyzed using relevant databases. Many cis-regulatory elements associated with phytohormones and abiotic stresses were identified in the promoter of MdSKP1-Like genes. Therefore, we performed the qrt-PCR to analyze the MdSKP1-Like gene family expression in different hormone treatments and abiotic stresses.

2. Results

2.1. Phylogenetic Tree Analysis of SKP1-Like Gene

The phylogenetic analysis of SKP1-Like proteins of six species were carried out using MEGA7. The results showed (Figure 1) that all SKP1-Like family members could be divided into three subfamilies. Among them, subfamily Ⅰ contained MdSKP1-Like2, MdSKP1-Like4 and MdSKP1-Like6; subfamily II contained MdSKP1-Like5, MdSKP1-Like8, MdSKP1-Like15, MdSKP1-Like16 and MdSKP1-Like17; and the rest of the MdSKP1-Like family members belonged to subfamily Ⅲ. Analysis of the subfamilies showed that subfamily I and subfamily III contained each of the six species studied, while subfamily II did not contain OsSKP1-Like members. Evolutionary relationships of subfamilies I and III indicate that members of the apple SKP1-Like family are more closely related to members of the strawberry SKP1-Like family members. Evolutionary relationships of subclade II indicate that apple SKP1-Like family members are more closely related to grape and strawberry SKP1-Like family members compared to other species. The above results suggest that plant SKP1-Likes have different characteristics in evolution, with apple SKP1-Like family members being more closely related to those of the dicotyledonous plants strawberry, grape, and Arabidopsis, and more distantly related to monocotyledonous plant rice and Ananas comosus SKP1-Like members in evolution.

2.2. Analysis of Physicochemical Properties and Chromosomal Localization

A total of 17 non-redundant MdSKP1-Like genes were identified from the whole apple genome, and their deduced amino acid sequences all contained typical Skp1 structural domains. According to their positions on chromosomes, the MdSKP1-Like genes were named MdSKP1-Like1-17 (Supplementary Table S1); the 17 MdSKP1-Like genes were unevenly distributed on eight chromosomes (Figure 2A). A total of four gene clusters were found, such as chromosomes 1, 8, 10 and 15. Four genes distributed on chromosome 10 were MdSKP1-Like11, MdSKP1-Like12, MdSKP1-Like13, and MdSKP1-Like14. There are four genes distributed on chromosome 1 MdSKP1-Like1, MdSKP1-Like2, MdSKP1-Like3, and MdSKP1-Like4. MdSKP1-Like16 and MdSKP1-Like17 were distributed on chromosome 15, while MdSKP1-Like5, MdSKP1-Like6, MdSKP1-Like7, and MdSKP1-Like15 were distributed on chromosomes 2, 4, 5 and 12, in that order. Also based on gene density, it was seen that MdSKP1-Like4, MdSKP1-Like8, and MdSKP1-Like16 were located in high-density regions. The average amino acid size, molecular weight/KD, isoelectric point, and instability index of proteins from members of subfamily Ⅰ are higher than those of members of the other two subfamilies (Figure 2B). The longest corresponding protein consisted of 356 amino acid residues (MdSKP1-Like2) and the shortest consisted of 108 amino acid residues (MdSKP1-Like1), the molecular weight (D) is 12147 (MdSKP1-Like1)~41130 (MdSKP1-Like2), and the isoelectric point (pI) is 4.27 (MdSKP1-Like16)~5.21 (MdSKP1-Like2) (Supplementary Table S1). The instability indices of these proteins ranged 30.86 (MdSKP1-Like12)~56.98 (MdSKP1-Like4), and we also found that these proteins are hydrophilic. The grand average of hydropathicity ranged −0.019 (MdSKP1-Like3)~−0.782 (MdSKP1-Like2), and the aliphatic index ranged 70.74 (MdSKP1-Like10)~110.14 (MdSKP1-Like3) (Supplementary Table S1).

2.3. The Subcellular Location Prediction and Secondary Structure Analysis

Subcellular localization of MdSKP1-Like genes analyses showed (Figure 3A) that the MdSKP1-Like genes were mainly located in the nuclear and cytoplasmic areas. Among them, MdSKP1-Like2, MdSKP1-Like4, and MdSKP1-Like6 were predicted to be predominantly distributed in the nucleus not in the lysosome. MdSKP-Like5, MdSKP1-Like8, MdSKP1-Like10, MdSKP1-Like14, MdSKP1-Like15, and MdSKP1-Like17 were predicted to have a high distribution in both the nucleus and cytoplasm, with most of the remaining members predicted to be predominantly distributed in the cytoplasm.
Apple SKP1-Like protein secondary structure (Figure 3B) showed that the secondary structure mainly consisted of alpha helix, extended strand, beta turn, and random coil. Alpha helix was mainly distributed between approximately 44.66 and 70.37%, the extended strand was mainly distributed between approximately 1.48 and 8.86%, the beta turn was mainly distributed between approximately 2.22 and 6.48%, and the random coil was mainly distributed between approximately 18.52 and 43.26%.

2.4. The MdSKP1-Like Gene Synteny Analysis

The MdSKP1-Like gene family synteny analysis results showed that three pairs of collinear relationships were found, namely MdSKP1-Like3/MdSKP1-Like6, MdSKP1-Like4/MdSKP1-Like10, and MdSKP1-Like2/MdSKP1-Like16 (Figure 4A). The results show that MdSKP1-Like genes may undergo amplification of family members through gene duplication during the evolutionary process. To further predict the phylogenetic components of the MdSKP1-Like family, we constructed interspecies collinear-relationship gene analysis maps for apple and Arabidopsis, rice, strawberry, and grape (Figure 4B). Among them, Apple had 14, 12, 10, and 1 homologous pairs of genes with strawberry, Arabidopsis, grape, and rice, respectively. Overall, MdSKP1-Like was more distantly related to OsSKP1-Like and more closely related to strawberry. Since they were more closely related genetically, it was hypothesized that MdSKP1-Like and FvSKP1-Like share some similarities in functional expression.

2.5. Gene Structure, Motif Composition and Structural Domain Analysis of MdSKP1-Like

Based on evolutionary tree relationships, the apple SKP1-Like gene family was divided into three groups (Figure 5). Group Ia consists of MdSKP1-Like5, MdSKP1-Like17, MdSKP1-Like7, MdSKP1-Like12, MdSKP1-Like16, MdSKP1-Like8, and MdSKP1-Like15, all of which contained conserved motif 1/2/4/6, and conserved structural domains skp1 and BTB-POZ-SKP1. Group IIb consisted of MdSKP1-Like4, MdSKP1-Like6, MdSKP1-Like14, MdSKP1-Like9, and MdSKP1-Like10, all of which contained motif 4 and the conserved structural domain skp1. The members of group IIIc consisted of MdSKP1-Like1, MdSKP1-Like2, MdSKP1-Like3, MdSKP1-Like11, and MdSKP1-Like13. Among them, MdSKP1-Like11 and MdSKP1-Like13 contain conserved motif 1/2/3/4/6, which were consistent with group Ia members. All members of the MdSKP1-Like family contain the conserved structural domain Skp1. As shown in Figure 5, the MdSKP1-Like gene’s introns and exon numbers were different in different subfamilies, with the exon numbers ranging from 1 to 14. MdSKP1-Like4 and MdSKP1-Like2 have the largest number of exons, 14 each, and MdSKP1-Like2 has the longest gene, about 64 kb, and MdSKP1-Like1 has the gene that was the shortest. The number and distribution of introns and exons within the same group are also different, and only a few of them have relatively high conservation. The structures of MdSKP1-Like7 and MdSKP1-Like12 in group Ia were similar; the structures of MdSKP1-Like11 and MdSKP1-Like13 in group IIIc were similar. It was hypothesized that MdSKP1-Like gene members with similar gene structures in each group perform the same functions among themselves.

2.6. Evolutionary Selection Pressure and Codon Usage Bias Analysis

We analyzed the Ka/Ks values of apple and Arabidopsis collinear relationship genes. The results showed that three gene pairs of MdSKP1-Like (Figure 6A) and six gene pairs of AtSKP1-Like had Ka/Ks values less than one (Figure 6B). This indicated that the SKP1-Like family members in apple and Arabidopsis were mainly under purifying selection. Among the analyzed codon usage deviations, the largest Nc value was 58.59 for MdSKP1-Like1 and the smallest Nc value was 39.53 for MdSKP1-Like12. The CAI values of MdSKP1-Like family members ranged from 0.15 to 0.37, and the frequencies of Fop ranged from 0.31 to 0.54 (Figure 6D). Compared to the values of A3s and T3s, we found larger values for GC3s and GCs for MdSKP1-Like family members (most values were around 0.5). The GC3s in the MdSKP1-Like family were positively related to CBI and Fop, but the correlation between T3S, A3S, CBI, and Fop in the MdSKP1-Like family was negative. (Figure 6E). RSCU is an assessment of the preference for the use of synonymous codons. Statistics revealed that GUC-encoded Val was used most frequently and with the greatest preference. CUA-encoded Leu was used least frequently and with the least preference (Figure. 6C). No preference was found for CAU and CAC-encoded His, AUG-encoded Met, CCA-encoded Pro, and UGG-encoded Trp, respectively.

2.7. Analysis of Protein Interactions of MdSKP1-Like Family Members

The protein interactions of MdSKP1-Like family members were predicted using the STRING website with Arabidopsis as the model plant. The results showed that the MdSKP1-Like8 homologous protein ASK2 (AT5G42190) (Supplementary Figure S1) interacted with AT1G76920, AT4G05460, galactose oxidase (ZTL), EIN3-binding F box protein 1 (EBF1), cullin1 (CUL1), regulator of cullins-1 (RBX1), corinsensitive 1 (COI1), and F-Box protein 7 (FBP7), etc (Figure 7A). Meanwhile, MdSKP1-Like11/12/15/16 were not involved in protein interactions, while the other members were involved in protein interactions with RBX1, FBP7, and other proteins interacted closely, indicating that the MdSKP1-Like family members exerted diverse functions in performing biological functions (Figure 7B), not only by having interactions among family members but also by interacting with many different proteins (especially the F-Box) at the same time.

2.8. Cis-Acting Elements Analysis of Apple MdSKP1-Like Gene Family

Using MdSKP1-Like gene upstream 2kb promoter to predict the cis-acting element. The results showed that the MdSKP1-Like gene promoter was related to hormones and stress (Figure 8). The hormone response element contains methyl jasmonate (CGTCA-motif, TGACG-motif), salicylic acid (TCA-element, as-1), auxin cis-elements (TGA-element), gibberellin (TATC-box), and abscisic acid (ABRE). We also identified many cis-regulatory elements associated with stress conditions, for example, regulatory elements involved in biotic and abiotic stresses in plants (MYB), MYB binding sites MBS and MYC. Other stress-specific cis-elements include drought-stress, salt-stress-sensitive reaction element (DRE), low-temperature reaction element (LTR), temperature-responsive stress reaction element (STRE), and anaerobic response element (ARE). Among them, the action elements related to salt stress, which were DRE and ABRE, the action element related to low temperature response (LTR), and the hormone element related to plant flowering (TATC-box). Notably, MdSKP1-Like8 was enriched for these four types of response elements simultaneously.

2.9. Analysis of MdSKP1-Like Gene Expression Based on GEO Database

Expression analyses of 17 MdSKP1-Like genes in seedling and different plant organs were performed by searching the GEO database. As shown, MdSKP1-Like8, MdSKP1-Like5, and MdSKP1-Like17 were expressed at high levels in all plant organs and seedling, and the remaining 14 genes were expressed at certain levels in different plant organs (Figure 9). Among them, MdSKP1-Like14 and MdSKP1-Like15 had a higher expression in flowers compared with other plant organs. Notably, other members of the MdSKP1-Like gene family, except MdSKP1-Like13, were expressed at higher levels in flowers, which, combined with the gibberellin regulatory element found in the upstream 2 KB of the promoter, predicts that MdSKP1-Like family members played a regulatory role in the growth and development of the flower.

2.10. Expression of MdSKP1-Like Gene in Apple Treated with Exogenous Hormones and Abiotic Stress Treatment

The above results indicated that the MdSKP1-Like gene may involve multiple stress-related cis-acting elements. After 24 h of treatment at 4 °C, all MdSKP1-Like members’ expression was below the control (Figure 10). After 24 h of PEG treatment, the expression of MdSKP1-Like2, MdSKP1-Like10, and MdSKP1-Like13 was higher than the control, and other members’ expression was lower than the control. The sensitivity of MdSKP1-Like genes to 24 h of NaCl treatment was higher than the control. Among them, the expression level of MdSKP1-Like9 was the highest. We also detected many elements related to hormone regulation at 2 kb upstream, so we could treat apple plants with different hormones, and the MdSKP1-Like gene expression differed significantly under different hormone treatments (Figure 10). Under MeJA treatment, the relative expression of MdSKP1-Like9 appeared to be downregulated and the rest of the genes were upregulated to some extent. After ABA treatment, the relative expression of MdSKP1-Like9 and MdSKP1-Like1 appeared to be downregulated and the rest of the genes were upregulated. Notably, the expression of MdSKP1-Like3 was highest under all three hormone treatments, respectively.

3. Discussion

3.1. Evolutionary Properties of the MdSKP1-Like Family

The SKP1-Like gene family was identified in many organisms, for example 21, 21, 31, and 19 members were identified from Cryptomeria hidrophila [21], Arabidopsis thaliana [12], rice [16], and tomato [13], respectively. The identification and systematic analysis of the SKP1-Like gene in apples is not clear. In this study, 17 SKP1-Like family members were identified from the apple. The physical- and chemical-property analysis shows that each member contains the conserved structural domain SKP1 in apples (Figure 5). Subcellular localization predicted that most MdSKP1-Like genes were located in the nucleus and cytoplasm, which corresponded to the SKP1-Like gene of Arabidopsis [27]; therefore, it was predicted that the MdSKP1-Like gene family was important for controlling the metabolism and genetic material of apple cells. The chromosomal localization map indicated that most MdSKP1-Like genes were located on the 10th chromosome (Figure 2A), in agreement with the results of previous studies on tomato [13]. All these results indicate that SKP1-like genes are conserved across the evolution of different species. This experiment also revealed that MdSKP1-Like genes located on chromosomes 1, 4, 10, and 15 have high homology (Figure 4A).
Previous experiments have shown that both the ASK1 gene in Arabidopsis and the OSK1 gene in rice originated from a single ancestor and that gene duplication provided the raw material for an evolutionary process that allowed these genes to perform similar functions [12,28]. Gene duplication is the main driver of gene family expansion, allowing the gene family to acquire new functions and evolve. Gene replication includes fragment replication, tandem replication and genome replication, with fragment replication being more beneficial for maintaining gene function [29]. The results showed that members of the MdSKP1-Like family undergo family-member amplification through fragmental replication during the evolutionary process. Previous studies divided the CaSKP1-Like family into three subfamilies based on genes with a single intron, lack of introns, and genes with a large number of introns at different locations [14]. In contrast, the MdSKP1-Like gene family was constructed from the monocotyledons and dicotyledons perspective in this study and was divided into three subfamilies, of which 3, 5, and 9 MdSKP1-like genes were sequentially distributed in subfamilies I, II, and III (Figure 1). The apple SKP1-Like family members were more closely related to dicotyledons (grape, strawberries) and more distantly related to monocotyledons (rice). The interspecies collinear relationship gene analysis also verified that MdSKP1-Like had more collinear relationships gene pairs with SKP1-Like family members of strawberry, and presumably the collinear members have similar biological functions, which also indicates to some extent that the apple was more closely related to SKP1-Like family members of strawberry (Figure 3B). These results may be because strawberry and apple belong to the same Rosaceae family.
The gene-selection pressure and codon bias analysis also contribute to the understanding of evolutionary relationships [30,31]. In this study, we calculated the intraspecific collinear relationship gene Ka/Ks ratios for Arabidopsis and apple, respectively, and most of them were less than one. The above results suggest that SKP1-Like family members in Arabidopsis and apple experience purifying selection effects. In addition, the codon bias analysis of MdSKP1-Like showed that GC3s in the MdSKP1-Like family were positively related to CBI and Fop, but the correlation between T3S, A3S, CBI, and Fop in the MdSKP1-Like family was negative (Figure 6). This suggests that the base type at position three of the synonymous codon of the MdSKP1-Like gene affects the magnitude of the degree of codon usage preference.

3.2. MdSKP1 May Interact with F-Box and May Respond to Hormone-Signaling Pathway

It was found that the protein interaction between SKP1 and F-Box was confirmed in an increasing number of species [27,32]. For example, ASK was able to interact with AtTLP9 and RCAR3 in Arabidopsis [33], and tomato SSK interacts with COI1 and TIR1 [13]. Furthermore, SKP1 has been shown to interact with F-Box in a non-SCF complex [34,35]. Results of protein interaction prediction in this study showed that most of the MdSKP1-Like family members may interact with FBP7, F-Box, and RBX1 proteins (Figure 7), but the specific mechanism of action needs to be further explored. The promoter analysis of MdSKP1-Like family members showed that MdSKP1-Like contains multiple hormone response elements. Among them, abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA) response elements were more abundant. It was suggested that ABA, MeJA, and SA may be important in the hormone response of the MdSKP1-Like family [23,36]. In SA or ABA treatments, nearly 50% of tomato SSK gene levels were upregulated, while under MeJA treatments most SSK genes were downregulated [13]. GMSK1 expression was upregulated in soybean under SA, ABA, and MeJA treatment after 48 h [18]. Following the same, we conducted different hormone treatments on apples, and the results showed that most MdSKP1-Like genes were upregulated after SA or ABA treatment, but MdSKP1-Like genes were also upregulated but to a lesser extent after MeJA treatment.
The Phylogenetic relationship between MdSKP1-like8 and ASK2 (AT5G42190) (Supplementary Figure S1) was the closest [11]. Based on the fact that the Arabidopsis ASK gene can regulate plant flowering [8], gibberellin was found to promote flowering in Arabidopsis by degrading DELLA [37], and the analysis of the upstream cis-acting elements of MdSKP-like8 revealed more gibberellin elements (TATC-box); it was speculated that MdSKP-like8 may regulate plant flowering. In combination with the previous study that found that ASK1 can regulate plant flowering and plant growth [38], it was hypothesized that these genes are important in regulating apple’s growth and development. It was also surprising to find high relative expression of most group Ia members in plant floral tissues (classified according to conserved motifs) (Figure 5), and more gibberellin-acting elements found in the node and presumably group Ia members may participate in the regulation of flower growth and development (Figure 9). Different tissue’s expression analyses showed that 17 MdSKP1-Like gene expressions were different in different plant tissues, and this difference in transcriptional expression could be attributed to the differentiation of cis-elements in promoters [39].

3.3. Structural Analysis of MdSKP1-Like Gene and Speculation on Its Function in Abiotic Stress Treatment

The distribution position of exons on the same group of amino acid sequences was highly variable, and there was no obvious pattern in sequence length. In this paper, 17 MdSKP1-Like genes were predicted to have nine conserved motifs (Figure 5), and similar results were also obtained in tomato SKP genes [13]. In the group Ia, the conserved motifs of all family members were consistent, indicating a high degree of conservation among the genes in the MdSKP1-Like group Ia. The results of the motif analysis showed that MdSKP1-Like2 and MdSKP1-Like4 contained two special motifs (motif 8, motif 9) at the C-terminus. Combined with a similar action element of 2000 bp upstream of MdSKP-Like2/4; these results imply that MdSKP1-Like2 and MdSKP1-Like4 may have specific functions that distinguish them from other members of the family. The number, length, and distribution of exons were not regular at the protein code level, but the degree of conservation among members was high at the protein level. Different members of the MdSKP1-Like family adopted different transcriptional and translational processes to finally form mature proteins with similar functions indicating that the degree of conservation at the genes of this family was not consistent with that at the protein level.
MdSKP1-Like cis-acting elements analyses indicated that these elements such as low-temperature and drought are present in large numbers, indicating their potential function in plant response to environmental stresses. Previously, many studies have also demonstrated the involvement of the SCF complex in the ABA pathway [40]. The receptor proteins of the SCF complex, DWA1 and DWA2, both of which exhibit high sensitivity to ABA and the ability of DWA1 and DWA2 to bind ABA insensitive 5 (ABI5) in vivo to degrade ABI5 [41]. Based on the discovery of a large number of ABRE cis-elements, it was hypothesized that the MdSKP1-Like gene may be involved in abiotic stress responses mediated by the ABA pathway. Extensive studies have demonstrated that SKP1 genes can enhance salt tolerance in pepper [19], and peony [17]. The relative expression of GmSKP1 in soybean reached its highest level at 24 h of NaCl treatment [18]. Likewise, under NaCl treatment, the relative expression of CaSKP1, CaSKP2, CaSKP5, CaSKP7, CaSKP8, CaSKP10, and CaSKP13 was upregulated in chickpea root systems [14]. In apple leaves treated with NaCl for 24 h, we found that MdSKP1-Like1, MdSKP1-Like5, and MdSKP1-Like9 were significantly upregulated. Under drought stress, all genes were downregulated, except for SSK5, SSK7, and SSK18 in tomato leaves. In chickpea roots and stems, we also found that most CaSKP genes were downregulated under drought stress [14]. We obtained similar results in apples, where most gene expression was downregulated after drought stress besides MdSKP1-Like2, MdSKP1-Like10, and MdSKP1-Like13. Most tomato SKP1-Like gene expression was downregulated under 4 °C temperature treatment [13]. Under 4 °C temperature treatment, all MdSKP1-Like gene expression was downregulated. These findings reveal the complexity of MdSKP1-like gene regulation and lay the foundation for further studies on the function of apple SKP1-like genes.

4. Materials and Methods

4.1. Plant Materials and Stress Treatment

The seedlings of the ‘Gala’ apple (Malus × domestica) were provided by the Fruit Seedling Research Laboratory of Gansu Agricultural University. After 30 d of succession culture, the seedlings were selected from healthy and uncontaminated cultures, and were successively treated with 10% PEG, at 4 °C low temperature, and with 150 mM NaCl. At the same time, plants with normal growth were used as the control. We treated the plants with each of the three hormones at 0.2 mM ABA, 5 mM SA, and 0.1 mM MeJA, and the same plants were treated with an equal distilled water as the control [18,42]. Three biological replicates were set up for each seedling’s treatment. Seedlings of uniform size were selected and the 4th~7th leaves from the base upward were quickly ground in liquid nitrogen to extract total RNA from the leaves using the CTAB method.

4.2. Identification and Characterization of Apple SKP1-Like Genes

The SKP1-Like gene family accession numbers of Oryza sativa L. and Arabidopsis were obtained from the relevant literature [11,12], and the corresponding full length CDS and full length genomic sequences were obtained in the tair databases (https://www.Arabidopsis.org, accessed on 16 September 2022) and rice genome databases (http://www.ricechip.org/, accessed on 20 September 2022). The obtained CDS sequences were compared using BLAST in the apple genome database (https://www.rosaceae.org/species/malus/all, accessed on 24 September 2022) [43], with the screening condition E ≤ 10−10. The screened genes were used in the apple genome database to search for the accession numbers, CDS sequences, protein sequences, and chromosomal location of predicted MdSKP1-Like gene family obtained using the homologous search. All predicted proteins were examined for structural integrity using the SMART (http://smart.embl-heidelberg.de/, accessed on 2 October 2022) online website [44], and sequences without the characteristic structural domains were removed to obtain the MdSKP1-Like genes. Protein’s physical and chemical properties were predicted using the online software ExPASy (https://web.expasy.org/protparam/, accessed on 4 October 2022) [45]. Using the online software CELLOV2.5 (http://cello.life.nctu.edu.tw/, accessed on 8 October 2022) [46], we predicted the subcellular localization of MdSKP1-Like genes.

4.3. Construction of a Phylogenetic Tree, Chromosome Localization, and Synteny Analysis

The ClustalX program was used to compare amino acids of SKP1-Like gene families of dicotyledonous plants such as apple, strawberry, grape, and Arabidopsis, and monocotyledonous plants such as rice and bromeliad to classify the MdSKP1-Like gene family according to monocotyledonous species. The evolutionary tree analysis software MEGA7.0 and the neighbor-joining method (NJ) were used to construct an evolutionary tree [47]. And phylogenetic tree embellishment was performed with Itol (https://itol.embl.de, accessed on 11 October 2022). MdSKP1-Like gene family location mapping was generated using TBtools (https://github.com/CJ-Chen/TBtools). Construction of synteny analysis maps between apples and other species using TBtools (version 1.108)

4.4. Construction of Gene Structure, Motif Sequence Analysis and Structural Domains

MdSKP1-Like gene’s exon and intron structures were obtained in the software TBtools (version 1.108) [48]. The MEME (http://meme.nbcr.ne-t/meme/cgi-bin/meme.cgi, accessed on 15 October 2022) set as any number of repetitions and the number of predicted motifs was 9 was used to analyze the protein conserved motifs of the MdSKP1-Like gene family. The data of the conserved structural domains of the apple SKP1-Like gene family were also obtained from the NCBI website (https://www.ncbi.nlm.nih.gov, accessed on 15 October 2022) [49] and mapped in the software TBtools.

4.5. Analysis of Selective Pressure and Codon Usage Index

The non-synonymous substitution rate and synonymous substitution rate of the MdSKP1-Like gene with co-linear relationships were calculated using the NG method of TBtools and mapped using Origin 2021. The CDS sequences of MdSKP1-Like were obtained and the codon usage characteristics of these genes were analyzed by CodonW software [50]. The main parameters included A3s, G3s, C3s and T3s (frequency of bases corresponding to synonymous codons at the third position), codon adaptation index (CAI), codon deviation index (CBI), optimal codon frequency (FOP), number of effective codons (Nc), number of third codons (G + C) (GC3s), number of genes (G + C) (GC), triple codons, and the correlations of these parameters were analyzed.

4.6. Analysis of MdSKP1-Like Member Protein Interactions

Select Arabidopsis as the model plant and set the highest confidence to 0.900, and STRING (https://string-db.org/, accessed on 15 March 2023) performed MdSKP1-Like family members protein interaction prediction [51]. It was further embellished with Cytoscape software and set the value to Betweenness (BC) for reconstruction.

4.7. Cis-Acting Elements of MdSKP1-Like and Gene Microarray Expression Analysis

The 2kb gene sequence upstream of the MdSKP1-Like was submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ accessed on 20 March 2023) and relevant gene action element data were obtained. Apple tissue expression data were downloaded from the GEO database for different tissue types [52]. Data sources are flower: M67; fruit: M74; leaf: M14; root: GD; stem: X8877; seedling: GD; and seed: X4442xX2596, so the SKP1-Like expression data were extracted according to its gene number. The obtained data were further organized using Excel software, and SKP1-Like expression analysis was completed using TBtools.

4.8. Quantitative Real-Time Fluorescence PCR (qRT-PCR) Analysis

The CDS sequences of MdSKP1-Like were submitted to the website of Bioengineering (Shanghai) Co. for online primer design (Table S2). cDNA synthesis was performed with the Prime Script RT reagent kit (Perfect Real Time) (TaKaRa). The expression of each gene was quantified using real-time fluorescence quantitative PCR (Mx3005p, Stratagene, La Jolla, CA, USA) using SYBR Green I kit (Takara Biomedical Technology Co., Ltd., Beijing, China) with the GAPDH gene as the internal reference gene. The relative expression of the genes was determined using 2−ΔΔCT [53].

5. Conclusions

Collectively, 17 MdSKP1-like gene members were identified, predicted to be mainly in the nucleus and cytoplasm. MdSKP1-Like gene expression in leaves varied greatly under different hormonal and abiotic stress treatments. qRT-PCR results also showed that MdSKP1-Like response to hormones and abiotic stress, under 4 °C treatment for 24 h, the expression of all MdSKP1-Like genes was downregulated. The expression of MdSKP1-Like2 and MdSKP1-Like10 genes was upregulated after both PEG and NaCl treatments for 24 h, respectively. In addition, the expression of most MdSKP1-Like genes was upregulated after 24 h of SA, ABA, and MeJA treatments. We also found that MdSKP1-Like3 was the most highly expressed gene in all three hormone treatments. These genes can be used as candidate genes for further functional studies of SKP1-Like genes involved in abiotic stresses and mediated phytohormone signaling pathways. Overall, these findings provide a comprehensive understanding of the SKP1-Like gene family in apples and contribute to the understanding of the role of SKP1-Like genes in apple growth, development, and adaptation to abiotic stresses.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms242216414/s1.

Author Contributions

Conceptualization, M.S. and P.W.; methodology, M.S. and P.W.; software, H.G.; validation, Z.M.; formal analysis, M.S.; writing—original draft preparation, M.S.; and writing—review and editing, J.M. and B.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Science and Technology Major Project of GansuProvince (22ZD2NA045), FuXi Foundation of Gansu Agricultural University (No. Ganfx-03J02).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be made available on request.

Conflicts of Interest

The authors declare that they have no conflict of interest associated with this work.

Abbreviations

E1sUbiquitin-activating enzyme
E2sUbiquitin-conjugating enzymes
E3sUbiquitin-protein ligases
SKP1S-phase kinase-associated protein 1
SKP1-likeS-phase kinase-associated protein 1-like
RBXRING Box; DUBs: Deubiquitinases
FBXF-Box; HRT1: Hairy-related transcription factor 1
ROC1Regulator of cullins
TIR1Transport inhibitor response 1
COI1CORONATINE INSENSITIVE 1
SLEEPY(SLY)S-linked F-box
ABAAbscisic acid
SASalicylic acid;
MeJAMethyl jasmonate
ATLPArabidopsis Tubby-like proteins
RCAR3ABA receptor proteins
DWA1 and DWA2DWD hypersensitive to ABA1 and 2

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Figure 1. Phylogenetic analysis of MdSKP1-Like gene family in apple. Dicots, Arabidopsis thaliana (At), Fragaria vesca (Fv), Malus domestica (Md), Vitis vinifera (Vv), and monocots Ananas comosus (Ac), Oryza sativa (Os) phylogenetic analysis of SKP1-Like proteins. The subfamilies were marked by a colorful background. Stars represent branching nodes of the evolutionary tree.
Figure 1. Phylogenetic analysis of MdSKP1-Like gene family in apple. Dicots, Arabidopsis thaliana (At), Fragaria vesca (Fv), Malus domestica (Md), Vitis vinifera (Vv), and monocots Ananas comosus (Ac), Oryza sativa (Os) phylogenetic analysis of SKP1-Like proteins. The subfamilies were marked by a colorful background. Stars represent branching nodes of the evolutionary tree.
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Figure 2. Distribution of MdSKP1-Like genes on different apple chromosomes and SKP1-Like protein physicochemical property violin diagram. (A) Chromosomes are indicated by colored bars. The position of the MdSKP1-Like gene is indicated next to it. The different colors represent gene density, where red indicates high-density regions and blue indicates low-density regions. (B) Violin box plots of amino acid size, molecular weight/KD, isoelectric point, instability index, aliphatic index, and grand average of hydropathicity for SKP1-Like proteins. Black dots represent outliers, and numbers with white dots represent averages.
Figure 2. Distribution of MdSKP1-Like genes on different apple chromosomes and SKP1-Like protein physicochemical property violin diagram. (A) Chromosomes are indicated by colored bars. The position of the MdSKP1-Like gene is indicated next to it. The different colors represent gene density, where red indicates high-density regions and blue indicates low-density regions. (B) Violin box plots of amino acid size, molecular weight/KD, isoelectric point, instability index, aliphatic index, and grand average of hydropathicity for SKP1-Like proteins. Black dots represent outliers, and numbers with white dots represent averages.
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Figure 3. The secondary structure- and subcellular-location prediction of MdSKP1-Like proteins. (A) Subcellular location prediction of MdSKP1-Like proteins. Purple for high numbers and blue for low numbers. (B) The secondary structure of MdSKP1-Like proteins. Different colors represent different secondary structures.
Figure 3. The secondary structure- and subcellular-location prediction of MdSKP1-Like proteins. (A) Subcellular location prediction of MdSKP1-Like proteins. Purple for high numbers and blue for low numbers. (B) The secondary structure of MdSKP1-Like proteins. Different colors represent different secondary structures.
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Figure 4. Collinearity relationship analysis of MdSKP1-Like gene. (A) Collinearity relationship of SKP1-Like family genes in apple. In the circles, the MdSKP1-Like gene family is labeled on the corresponding chromosome, and the represented collinearity gene pair between the MdSKP1-Like genes is indicated by the purple curve. The outermost circle with color and the second outermost circle are expressions of gene density. The purple color has a higher density, and the blue color has the lowest density. (B) Collinearity relationships of MdSKP1-Like genes between apples and four representative plant species. The gray line in the background indicates apples and their neighboring blocks in the plant genome, while the black line highlights the collinearity SKP1-Like gene pairs.
Figure 4. Collinearity relationship analysis of MdSKP1-Like gene. (A) Collinearity relationship of SKP1-Like family genes in apple. In the circles, the MdSKP1-Like gene family is labeled on the corresponding chromosome, and the represented collinearity gene pair between the MdSKP1-Like genes is indicated by the purple curve. The outermost circle with color and the second outermost circle are expressions of gene density. The purple color has a higher density, and the blue color has the lowest density. (B) Collinearity relationships of MdSKP1-Like genes between apples and four representative plant species. The gray line in the background indicates apples and their neighboring blocks in the plant genome, while the black line highlights the collinearity SKP1-Like gene pairs.
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Figure 5. MdSKP1-Like gene structure, motif, and structural domain analyses. Subclade Ia is highlighted in purple; subclade IIb is highlighted in blue; and subclade IIIc is highlighted in pink. (Left) Conserved motifs; different motifs are marked with different colors; and the numbers above represent different motifs. (Middle) Structural domain map; (Right) the exon-intron structure of MdSKP1-Like gene; and the number represents quantities.
Figure 5. MdSKP1-Like gene structure, motif, and structural domain analyses. Subclade Ia is highlighted in purple; subclade IIb is highlighted in blue; and subclade IIIc is highlighted in pink. (Left) Conserved motifs; different motifs are marked with different colors; and the numbers above represent different motifs. (Middle) Structural domain map; (Right) the exon-intron structure of MdSKP1-Like gene; and the number represents quantities.
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Figure 6. MdSKP1-Like gene evolutionary selection pressure and codon usage bias analyses. (A) Ka/Ks analysis of SKP1-Like collinearity relationship gene pairs. (B) Ka/Ks analysis of collinearity relationship gene pairs SKP1-Like genes in Arabidopsis. (C) Relative synonymous codon usage (RSCU). (D) Synonymous codon preference and correlation analyses of MdSKP1-Like gene. Different colors indicate the magnitude of correlation coefficients; the larger the correlation coefficients are in purple, and the smaller numbers are in blue. U3s, C3s, A3s, G3s, and T3s indicate the U, C, A, and T of the codon third site, respectively. G + C base composition content. CAI, codon adaptation index. CBI, codon preference index. FOP, frequency of optimal codon usage. NC: effective codon number. GC content of the third position of GC3s synonymous codon. GC: GC content. (E) Correlation analysis of MdSKP1-Like gene codon. Purple indicates a positive correlation; blue indicates a negative correlation; and white indicates no correlation.
Figure 6. MdSKP1-Like gene evolutionary selection pressure and codon usage bias analyses. (A) Ka/Ks analysis of SKP1-Like collinearity relationship gene pairs. (B) Ka/Ks analysis of collinearity relationship gene pairs SKP1-Like genes in Arabidopsis. (C) Relative synonymous codon usage (RSCU). (D) Synonymous codon preference and correlation analyses of MdSKP1-Like gene. Different colors indicate the magnitude of correlation coefficients; the larger the correlation coefficients are in purple, and the smaller numbers are in blue. U3s, C3s, A3s, G3s, and T3s indicate the U, C, A, and T of the codon third site, respectively. G + C base composition content. CAI, codon adaptation index. CBI, codon preference index. FOP, frequency of optimal codon usage. NC: effective codon number. GC content of the third position of GC3s synonymous codon. GC: GC content. (E) Correlation analysis of MdSKP1-Like gene codon. Purple indicates a positive correlation; blue indicates a negative correlation; and white indicates no correlation.
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Figure 7. Protein interaction analysis of the SKP1-Like. (A) Interaction analysis of ASK2, a homolog of SKP1-Like8, with other proteins. Purple color represents proteins with tighter interprotein interactions, and blue color is the opposite. (B) Protein interaction analysis of the SKP1-Like in apple. Blue color represents MdSKP1-like protein members and purple color represents protein members that interact with them.
Figure 7. Protein interaction analysis of the SKP1-Like. (A) Interaction analysis of ASK2, a homolog of SKP1-Like8, with other proteins. Purple color represents proteins with tighter interprotein interactions, and blue color is the opposite. (B) Protein interaction analysis of the SKP1-Like in apple. Blue color represents MdSKP1-like protein members and purple color represents protein members that interact with them.
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Figure 8. The first 2000 bp of cis-acting elements of 17 MdSKP1-Like genes. Different elements are labeled with different colors. (A) Abiotic stress-regulating elements predominate. (B) Major phytohormone action elements. Numbers represent the number of cis-acting elements.
Figure 8. The first 2000 bp of cis-acting elements of 17 MdSKP1-Like genes. Different elements are labeled with different colors. (A) Abiotic stress-regulating elements predominate. (B) Major phytohormone action elements. Numbers represent the number of cis-acting elements.
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Figure 9. Hierarchical clustering of the expression profiles of the MdSKP1-Like gene in seedling and different organs in apple. (A) Heat map of the expression of MdSKP1-Like genes in seedling and different organs of apple. (B) Cluster analysis of heat map data of MdSKP1-Like gene based on GEO databases.. Purple and blue represent upregulated or downregulated expression levels, respectively.
Figure 9. Hierarchical clustering of the expression profiles of the MdSKP1-Like gene in seedling and different organs in apple. (A) Heat map of the expression of MdSKP1-Like genes in seedling and different organs of apple. (B) Cluster analysis of heat map data of MdSKP1-Like gene based on GEO databases.. Purple and blue represent upregulated or downregulated expression levels, respectively.
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Figure 10. Expression levels of MdSKP1-Like gene in abiotic stresses and phytohormones treatments. Expression levels of MdSKP1-Like gene in NaCl, 4 °C, PEG, MeJA, ABA, and SA phytohormones treatments. Statistical analysis was performed using one-way ANOVA and Tukey’s honestly significant difference (HSD) test. The expression level of the control group that was not stressed has a value of 1. Black error lines represent the mean ± SE of three biological replicates. Different letters denote significant differences, whereas the same lowercase letters indicate no statistical difference (p < 0.05).
Figure 10. Expression levels of MdSKP1-Like gene in abiotic stresses and phytohormones treatments. Expression levels of MdSKP1-Like gene in NaCl, 4 °C, PEG, MeJA, ABA, and SA phytohormones treatments. Statistical analysis was performed using one-way ANOVA and Tukey’s honestly significant difference (HSD) test. The expression level of the control group that was not stressed has a value of 1. Black error lines represent the mean ± SE of three biological replicates. Different letters denote significant differences, whereas the same lowercase letters indicate no statistical difference (p < 0.05).
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MDPI and ACS Style

Shao, M.; Wang, P.; Gou, H.; Ma, Z.; Chen, B.; Mao, J. Identification and Expression Analysis of the SKP1-Like Gene Family under Phytohormone and Abiotic Stresses in Apple (Malus domestica). Int. J. Mol. Sci. 2023, 24, 16414. https://doi.org/10.3390/ijms242216414

AMA Style

Shao M, Wang P, Gou H, Ma Z, Chen B, Mao J. Identification and Expression Analysis of the SKP1-Like Gene Family under Phytohormone and Abiotic Stresses in Apple (Malus domestica). International Journal of Molecular Sciences. 2023; 24(22):16414. https://doi.org/10.3390/ijms242216414

Chicago/Turabian Style

Shao, Miao, Ping Wang, Huimin Gou, Zonghuan Ma, Baihong Chen, and Juan Mao. 2023. "Identification and Expression Analysis of the SKP1-Like Gene Family under Phytohormone and Abiotic Stresses in Apple (Malus domestica)" International Journal of Molecular Sciences 24, no. 22: 16414. https://doi.org/10.3390/ijms242216414

APA Style

Shao, M., Wang, P., Gou, H., Ma, Z., Chen, B., & Mao, J. (2023). Identification and Expression Analysis of the SKP1-Like Gene Family under Phytohormone and Abiotic Stresses in Apple (Malus domestica). International Journal of Molecular Sciences, 24(22), 16414. https://doi.org/10.3390/ijms242216414

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