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Article

Identification of Ascorbate Oxidase Genes and Their Response to Cold Stress in Citrus sinensis

by
Xiaoyong Xu
1,*,
Xingchen Miao
1,
Naiyi Deng
1,
Mengge Liang
2,
Lun Wang
1,
Lijuan Jiang
1 and
Shaohua Zeng
3
1
College of Horticulture and Landscape Architecture, Yangzhou University, Yangzhou 225009, China
2
College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan 430070, China
3
Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(9), 1643; https://doi.org/10.3390/agriculture14091643
Submission received: 24 July 2024 / Revised: 17 September 2024 / Accepted: 18 September 2024 / Published: 19 September 2024
(This article belongs to the Special Issue Abiotic Stress Responses in Horticultural Crops)
Browse Figures
Versions Notes

Abstract

:
Ascorbate oxidase (AAO) plays an important role in maintaining cellular redox homeostasis, thereby influencing plant growth, development, and responses to both biotic and abiotic stresses. However, there has been no systematic characterization of AAO genes in Citrus, especially their roles in response to cold stress. In the present study, nine AAO genes were identified in C. sinensis through bioinformatics analyses, exhibiting uneven distribution across four chromosomes. All CsAAOs possessed three conserved domains and were predicted to localize in the apoplast. The CsAAO gene family displayed varied intron–exon patterns. Phylogenetic analysis categorized the CsAAO family into three main clades (Clade A–C), suggesting distinct biological functions. Collinearity and Ka/Ks analysis revealed three duplicate gene pairs within the CsAAO gene family, with all duplicated CsAAOs primarily evolving under purifying selection. Analysis of cis-acting elements showed the presence of multiple hormone response elements and stress response elements within the CsAAO promoters. The computational analysis of microRNA target transcripts suggested that CsAAO9 may be a target of csi-miR156. RNA-Seq data demonstrated high expression levels of CsAAOs in roots and young fruits, while qRT-PCR analysis showed significant upregulation of six CsAAOs in response to cold treatment. Furthermore, the activities of CsAAOs exhibited a pattern of initial decrease followed by an increase after exposure to low temperatures. These findings offer important insights into the role of CsAAOs in response to cold stress. Furthermore, AAOs could be target genes for breeding crops with better cold resistance.

1. Introduction

Ascorbate oxidases (EC 1.10.3.3, AAOs) are classified as multi-copper oxidases and are found exclusively in plants and fungi [1]. AAOs are primarily located in the apoplast and their primary function is to facilitate the oxidation of ascorbate acid (AsA, reduced ascorbate) to monodehydroascorbate (MDHA) while simultaneously reducing molecular oxygen to water. MDHA undergoes spontaneous conversion to dehydroascorbate (DHA, oxidized ascorbate), which is then transported to the cytoplasm, where it is converted back to AsA through the ascorbate–glutathione (AsA-GSH) cycle [2]. Existing research indicates that AAOs play a crucial role in regulating the apoplastic redox state, thereby maintaining balanced levels of reactive oxygen species [3,4,5]. Additionally, AAOs may be involved in the processes of cell expansion and the loosening of cell walls in melon fruit [3,6]. Therefore, AAOs participate in the maintenance of cellular redox homeostasis, thereby exerting an influence on plant growth and development, as well as modulating responses to biotic and abiotic stresses [4]. It is increasingly evident that AAOs are being considered potential candidates for enhancing crop stress tolerance [3,7].
Since AAOs were first described in cabbage leaf by Szent-Györgyi [8], it has been extensively investigated [1,3]. Early studies of AAOs primarily examined the enzyme’s biochemical properties before expanding to explore its biological function. Of particular significance is the isolation of AAO cDNA from cucumber fruit tissues in 1989, leading to the subsequent cloning and identification of numerous AAO genes in various plant species [9,10,11,12]. In plants, AAOs are encoded by multiple genes, and some AAO gene family members have been identified and characterized in various plant species, including three members in Arabidopsis, five in rice, eight in cotton, four in maize, fourteen in bread wheat, seven in soybean, twelve in sugar beet, six in sorghum, and sixteen in Ammopiptanthus nanus [13,14,15,16]. Based on current understanding, the expression levels and enzymatic activity of AAOs exhibit notable variations across diverse plant species and tissues. For instance, Cucurbitaceae plants demonstrate elevated AAO activity in comparison to Solanaceae species. In tobacco, AAO genes are notably expressed in young and actively growing tissues, including the upper leaf, upper stem, and root [11]. In melon (Cucumis melo), the CmAO4 gene is more abundant in vegetative tissues, whereas the CmAO1 gene is predominantly expressed in fruit and floral tissues [17]. Additionally, in bread wheat (Triticum aestivum), the majority of TaAAOs exhibit high expression in root tissues [18]. These findings suggest that AAOs play crucial and varied functional roles across different tissue types.
The role of AAOs in environmental stress responses has garnered a growing interest, as AAOs serve as a key regulator of redox status in the apoplast. Typically, in transgenic plants, AAOs demonstrate a negative regulatory effect on drought and salt stress tolerance, as well as on disease resistance to fungal pathogens. For instance, in rice and sugar beet, the expression of AAOs decreased significantly under drought and salt treatments [13,15]. Notably, AAO antisense plants exhibited greater tolerance to drought or salt stress compared to wildtype plants in tomato [19,20]. Additionally, tobacco plants overexpressing AAOs showed increased susceptibility to fungal pathogen infection [21]. Interestingly, AAOs exhibit positive regulation of cold stress and viral resistance in transgenic plants. In A. nanus, the expression of three AAO genes was upregulated after seven days of cold stress, and overexpression of AnAAO5 improved the cold stress tolerance of Arabidopsis plants [16]. In tobacco or rice, virus infection typically induces the expression of AAOs, while silencing AAOs has been shown to significantly diminish disease resistance [22,23]. This discrepancy may be attributed to alterations in ascorbic acid levels resulting from changes in AAO expression [3].
Citrus, particularly sweet orange (C. sinensis), is a significant and extensively cultivated fruit crop globally. Despite its widespread cultivation, low temperature stress significantly impacts the growth, development, and distribution of Citrus, posing challenges to the Citrus industry [24,25]. For example, the cold waves experienced in 2016 and 2021 led to differing levels of economic losses within China’s citrus industry. Thus, the identification of key genes responsive to low temperatures is essential for the development of cold-resistant Citrus varieties [26,27,28,29]. While the AAO gene is known to play a crucial role in abiotic stress responses, its specific involvement in Citrus low-temperature stress remains unclear. In the present study, nine AAO genes were identified from the C. sinensis genome and their physicochemical properties, chromosomal localization, gene structures, phylogenetic relationships, duplication events, and cis-acting elements were analyzed. Subsequently, the expression profiles of CsAAO genes in various tissues and their response to cold stress were examined using public RNA-seq data and quantitative real-time PCR (qRT-PCR). Finally, the AAO activity of C. sinensis leaves was investigated after cold treatment. Overall, the findings of this study offer valuable insights for future functional analysis of CsAAO genes in the context of cold stress response.

2. Materials and Methods

2.1. Genome-Wide Identification of the AAO Gene Family in Citrus sinensis

For the purpose of identifying Citrus AAO genes, the protein sequences and genome of Citrus were obtained from the Citrus Pan-genome to Breeding Database. BLASTP analysis was conducted using three Arabidopsis AAO protein sequences sourced from The Arabidopsis Information Resource, along with the Hidden Markov Model (HMM) profile of the three Cu-oxidase domains (PF00394, PF07731, and PF07732) obtained from the pfam database as the queries. The putative CsAAOs were subsequently validated using the Pfam and SMART databases to identify conserved domains. The CsAAOs were designated CsAAO1–9 based on their chromosomal location, with the chromosomal location map of these genes visualized using TBtools software (https://github.com/CJ-Chen/TBtools/ accessed on 6 March 2023) [30].

2.2. Physicochemical Properties Analysis

The molecular weight, isoelectric point, instability index, and grand average of hydropathicity of each CsAAO were predicted using the online software ExPASy (http://web.expasy.org/protparam/, accessed on 6 March 2023). The subcellular localization of CsAAOs was predicted by the online software CELLO (v.2.5, http://cello.life.nctu.edu.tw/, accessed on 6 March 2023) [31].

2.3. Gene Structure and Conserved Motif Analysis

The exon–intron distribution pattern of CsAAO genes was displayed based on the genome annotation file using TBtools software [30]. The MEME database (https://www.kaggle.com/datasets/utkarshx27/meme-database, accessed on 6 March 2023) was utilized to predict conserved motifs in CsAAO proteins, with a maximum number of 6 motifs and default parameters.

2.4. Phylogenetic Tree, Ka/Ks, and Collinearity Analysis

Phylogenetic trees were generated utilizing the Neighbor-joining algorithm in MEGA X software (https://www.megasoftware.net/, accessed 10 March 2023). The nucleotide substitution rates for nonsynonymous (Ka) and synonymous (Ks) mutations, as well as the Ka/Ks ratios of homologous CsAAO gene pairs, were determined using the simple Ka/Ks calculator in TBtools. Collinearity analysis between the genomes of Citrus and rice/Arabidopsis was conducted using the Multiple Systeny Plot in TBtools.

2.5. Prediction of Cis-Acting Elements and microRNAs Targeting CsAAO Genes

The cis-acting regulatory elements within the 2000 bp region upstream of the transcription start site of CsAAOs were predicted through the PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed 12 March 2023) [32]. The Citrus microRNA sequences retrieved from the miRBase database were subjected to analysis for miRNA target site presence in CsAAOs by the psRNATarget server [33], with stringent parameters set at a maximum expectation value of 3.5 and default settings for all other parameters.

2.6. Expression Profiles of CsLACs in Various Tissues

The FPKM values for CsAAO genes were downloaded from the Citrus Pan-genome to Breeding Database (http://citrus.hzau.edu.cn/, accessed on 12 May 2023). Heatmaps illustrating the expression profiles of CsAAOs were generated using TBtools.

2.7. Plant Materials and Low Temperature Treatments

Sweet orange plants (C. sinensis) were cultivated in a greenhouse under controlled conditions of 25 °C and 60% humidity, with a photoperiod of 16 h of light and 8 h of darkness. For the low temperature treatment, one-year-old sweet orange seedlings were subjected to an immediate temperature reduction to 4 °C for varying duration of 0, 2, 4, 6, 8, and 12 h. Following treatment, leaf samples were promptly frozen in liquid nitrogen and stored at −80 °C until RNA extraction. AAO activities were assessed using commercially available assay kits (Shanghai Shangbao Biotechnology Ltd., Shanghai, China).

2.8. RNA Extraction and Expressing Profile Analysis

Total RNA was isolated utilizing RNA Isolation Reagent (Nanjing Nuoweizan Biotechnology Co., Ltd., Nanjing, China) according to the manufacturer’s protocol. RNA quality was assessed through gel electrophoresis and a bioanalyzer (Agilent2100, Beijing, China). Subsequently, first strand cDNA synthesis was carried out using HiScript First Strand cDNA Synthesis Kit (Nanjing Nuoweizan Biotechnology Co., Ltd., China) following the manufacturer’s guidelines. Data analysis was conducted with β-actin expression serving as a reference. The gene-specific primers utilized for qRT-PCR analysis are detailed in Supplementary Table S1. The qRT-PCR experiments were conducted using the CFX96 real-time PCR machine manufactured by BIO-RAD (Hercules, CA, USA). The reaction conditions and protocol for qPCR were consistent with those described in our previous publication [27]. The 2−ΔΔCT Ct method was employed to determine the relative gene expression levels across the samples [34]. For each experiment, three biological replicates were performed.

3. Results

3.1. Identification, Physicochemical Properties, and Chromosomal Location of the CsAAO Family

Based on BLASTP and HMMER searches, a total of nine candidate CsAAO genes were identified from C. sinensis genome (v3.0). These putative CsAAOs were designated as CsAAO1 to CsAAO9 according to their chromosomal positioning. As shown in Figure 1, nine CsAAO genes were randomly anchored on four chromosomes (chr1, chr2, chr3, and chr8). The physicochemical characteristics of CsAAO genes were analyzed, revealing variations in the amino acid length of CsAAO proteins ranging from 538 (CsAAO5) to 586 (CsAAO1) and calculated molecular weights from 59.52 kDa to 66.16 kDa (Table 1). The isoelectric point values fell within a range of 7.37 (CsAAO6) and 9.37 (CsAAO4). Among the CsAAO proteins, only CsAAO1 and CsAAO4 exhibited instability, with instability index (II) values exceeding 40. The grand average of hydropathicity (GRAVY) values for all CsAAO proteins was negative, indicating hydrophilicity. Additionally, subcellular localization prediction using CELLO v.2.5 software suggested that all CsAAO proteins were extracellularly localized.

3.2. Analysis of Conserved Motifs, Domain Composition, and Gene Structure of CsAAOs

The comparison of conserved motifs, domain composition, and gene structure among members of a gene family is widely recognized as a valuable approach for gaining insights into their functional characteristics. In the present study, six conserved motifs were identified in CsAAO proteins, as illustrated in Figure 2. It is noteworthy that CsAAO1 and CsAAO6 within the same group were found to lack motif 4 (Supplementary Figure S1), whereas all other CsAAO proteins exhibited the presence of all six motifs (Figure 2a,b). Additionally, Figure 2c demonstrates the presence of Cu-oxidase, Cu-oxidase_2, and Cu-oxidase_3 domains in all CsAAO proteins. Examination of the gene structures of CsAAO revealed variations in the number of exons, ranging from 2 to 8 (Figure 2d). Interestingly, the size of CsAAO genes appeared to be influenced by the size of introns. In summary, certain CsAAO genes within the same group exhibit similar gene structures.

3.3. Phylogenetic Analysis of AAO Proteins in Different Species

Phylogenetic analysis was conducted using 17 AAO predicted proteins from Citrus, Arabidopsis, and rice to construct an evolutionary tree (Figure 3). The resulting phylogenetic tree revealed that the AAOs were grouped into three distinct clades, with CsAAOs present in each clade. Specifically, CsAAO6 was assigned to Clade A, CsAAO1 to Clade B, and CsAAO2, CsAAO3, CsAAO4, CsAAO5, CsAAO7, CsAAO8, and CsAAO9 to Clade C.

3.4. Gene Duplication and Genome Synteny Analysis of AAO Family Genes

In order to examine the gene duplication events of the AAO family, a collinearity analysis was conducted. Within the CsAAO gene family, the analysis revealed the presence of two segmental duplication gene pairs (CsAAO4—CsAAO5, CsAAO8—CsAAO9) and one tandem duplication gene pair (CsAAO2—CsAAO3) within the C. sinensis genome (Figure 1), suggesting that gene duplication events have facilitated the expansion and evolution of the CsAAO family. Additionally, the Ka/Ks ratio for each duplicated gene pair was found to be less than 0.5, indicating that all duplicated genes have been subjected to strong purifying selection (Table 2). Comparing the CsAAO family with rice and Arabidopsis AAO family revealed a high homology between C. sinensis and Arabidopsis genes, suggesting similar functions. The collinear region between C. sinensis and Arabidopsis is greater than that with rice, indicating closer orthologs between C. sinensis and Arabidopsis (Figure 4).

3.5. Cis-Acting Element and MicroRNA Target Site Analysis of CsAAOs

Cis-acting elements have been identified as crucial components in the control of gene expression [35]. In order to gain a deeper insight into the function of CsAAO genes and the mechanisms of transcriptional regulation, the cis-acting elements located within the 2000 base pair upstream regions of the translation initiation site were analyzed for nine CsAAO genes utilizing the PlantCARE software (Figure 5). The examination of cis-acting elements indicated that the promoter regions of CsAAO genes encompass 39 distinct types of cis-acting elements, which can be classified into three primary categories: development elements, hormone response elements, and stress response elements. Notably, stress response elements were found to be the most prevalent, comprising a total of 25 occurrences. Development elements primarily include meristem-specific elements (CAT-box), circadian regulation elements (circadian), and endosperm expression regulatory elements (GCN4_motif). Hormone response elements mainly consist of abscisic acid-responsive elements (ABREs), methyl jasmonate-responsive elements (CGTCA-motif), and salicylic acid-responsive elements (TCA-elements). Stress response elements mainly encompass light-responsive elements (Box 4, G-Box, etc.), anaerobic induction elements (AREs), and low-temperature-responsive (LTR) elements. Of note, the promoter region of some CsAAO genes contains unique cis-acting elements, such as wound-responsive elements (WUN-motif) in the CsAAO1 promoter and gibberellin-responsive elements (TATC-box) in the CsAAO6 promoter.
MicroRNAs (miRNAs) are a group of small endogenous non-coding RNAs, typically 20–24 nucleotides in length, known for their ability to downregulate gene expression post-transcriptionally through mechanisms such as direct transcript cleavage or translational inhibition. This regulatory function of miRNAs has been shown to impact various aspects of plant biology, including growth, development, and response to stress [36]. In this study, all miRNAs identified in Citrus were utilized as query sequences to predict potential target sites on CsAAO genes using the psRNATarget tool with stringent parameters. The analysis revealed that two specific Citrus miRNAs (csi-miR156h/i) are likely regulators of the CsAAO9 gene (Table 3).

3.6. Gene Expression Analysis of CsAAOs in Different Tissues

In order to elucidate the potential functions of CsAAO genes, an analysis was conducted on the available RNA-seq data from the Citrus Pan-genome to Breeding Database. The findings revealed that CsAAO gene expression displays tissue specificity, with CsAAO2 and CsAAO6 exhibiting notably high expression levels in various tested tissues such as leaves, seeds, callus, roots, early-stage ovules, late-stage ovules, young fruit flesh, and mature fruit flesh (Figure 6). Conversely, CsAAO7 was not detected in any of the eight tissues examined. Noteworthy is the observation that CsAAO3 was exclusively expressed in roots. Overall, the nine CsAAO genes demonstrated higher expression levels in roots and young fruit tissues.

3.7. Analysis of CsAAOs Expression Patterns and Enzymatic Activities under Cold Stress

The AAO gene family is known to be involved in abiotic stress responses. Therefore, quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to assess the transcript levels of AAO in response to low temperature treatment in order to investigate the ability of the CsAAO family to respond to cold stress. As depicted in Figure 7, the expression patterns of the CsAAO1, CsAAO3, CsAAO5, and CsAAO7 genes exhibit an initial increase followed by a decrease, whereas the CsAAO2, CsAAO4, CsAAO6, and CsAAO8 genes demonstrate a decrease followed by an increase. CsAAOs exhibiting a marked decline in expression from 0 to 2 h may experience a transient transcriptional repression as a consequence of the cold shock. The expression of the CsAAO9 gene shows a consistent upward trend. Noteworthy is the substantial upregulation of CsAAO1 by 42.4-fold at 2 h, CsAAO7 by 130.9-fold at 8 h, and CsAAO9 by 47.3-fold at 12 h compared to the control group. The observations revealed that CsAAO1, CsAAO5, CsAAO7, and CsAAO9 were implicated in the cold stress response of C. sinensis. Additionally, the activities of the CsAAOs demonstrated a pattern of initial decline, followed by an increase upon exposure to low temperatures (Figure 8).

4. Discussion

In the current work, we identified nine AAO genes from C. sinensis by bioinformatics analyses. The number of AAO gene family members varies from 3 to 16 among different plant species [13,14,15,16]. After analyzing the AAO gene family members across various species and their respective genome sizes, it was determined that there is no significant correlation between genome size and the number of AAO gene family members [18]. Additionally, there is no significant difference in the number of AAO gene members between monocotyledonous and dicotyledonous plants. Subcellular localization predicted that all CsAAO proteins were localized in the apoplast (Table 1). These findings align with previous research [16,18], indicating the proteins’ potential involvement in maintaining cellular redox homeostasis. Examination of conserved motifs, domain composition, and gene structure of CsAAOs revealed that the CsAAO gene family exhibited a notable degree of conservation throughout the course of evolution (Figure 2). Notably, the conserved domains/motifs within the AAO gene family members exhibit high similarity in five plant species including rice, Arabidopsis, soybean, maize, and sorghum [13]. Thereby, the AAO gene family may be an ancient and evolutionarily conserved gene family in plants.
To compare the function and evolutionary relationships of CsAAO proteins in dicots and monocots, amino acid sequences from rice, Arabidopsis, and C. sinensis AAOs were used to create a phylogenetic tree (Figure 3). The results showed that AAOs from C. sinensis, Arabidopsis, and rice grouped into three clades, supporting previous findings [13,14]. The presence of the same clades in all three species suggests that AAO genes existed before the split between monocots and dicots. Furthermore, it was observed that seven CsAAOs from C. sinensis and OsAAO3 from the monocot model plant rice were clustered together in the same phylogenetic branch. The OsAAO3 gene was responsive to salinity and drought stresses [13]. Consequently, it is hypothesized that the seven CsAAOs may share a similar function with OsAAO3. However, additional research is required to validate this assertion.
Analysis of gene collinearity within and between species offers valuable information on genome evolution, conservation of gene function, and evolutionary connections [37]. In this study, we have identified three putative C. sinensis paralogs (Cs-Cs), thirteen orthologs shared between C. sinensis and Arabidopsis thaliana (Cs-At), and two orthologs shared between C. sinensis and Oryza sativa (Cs-Os) (Figure 1 and Figure 4). In Glycine max, three AAOs duplicated tandemly and four AAOs duplicated segmentally [13]; in Ammopiptanthus nanus, four AAOs duplicated tandemly and eight AAOs duplicated segmentally [16]; in Gossypium hirsutum, two AAOs duplicated tandemly and six AAOs duplicated segmentally [14]. These findings suggest that gene duplication events, particularly segmental duplication, played a significant role in the evolutionary development and proliferation of the AAO gene family in plant species [38]. Additionally, Ka/Ks analysis indicated that the duplicated genes of CsAAOs may have been subjected to significant purifying selection pressure (Table 2), a finding consistent with previous studies on Ammopiptanthus nanus and Gossypium hirsutum [14,16].
MicroRNAs have been demonstrated to be significant regulators of gene expression post-transcriptionally. A recent study identified miR528 as a negative regulator of viral resistance in rice by targeting AAO messenger RNA [23]. Additionally, miR4415 was predicted to target AnAO5 and AnAO11 in Ammopiptanthus nanus [16]. Our findings suggest that csi-miR156 may target CsAAO9, although experimental validation is required to confirm this prediction.
PlantCARE analysis revealed multiple cis-acting elements in the promoters of CsAAO genes, categorized into development, hormone response, and stress response elements (Figure 5). This distribution pattern is consistent with other plant species [14,16], suggesting that AAO genes play a role in multifarious physiological procedures. The predominant cis-acting elements identified in this study were stress response elements, with a total of 25 instances observed. The stress response elements include light-, anaerobic induction-, low-temperature-, drought-, wound-, and defense/stress-responsive elements. These cis-acting elements help CsAAOs respond to oxidative stress from adverse conditions, aiding in maintaining extracellular redox balance. The promoter regions of all CsAAO genes, with the exception of CsAAO9, contain anaerobic induction elements. It is well established that anaerobic induction elements are typically activated in roots following water excess and during seed germination [39]. Our observations further indicate that the majority of CsAAOs exhibit high expression levels in roots (Figure 6). These findings suggest that CsAAOs may play a role in conferring anaerobic tolerance in roots. Notably, the promoter regions of certain CsAAO genes have unique cis-acting elements, like wound-responsive elements (WUN-motif) in CsAAO1 and gibberellin-responsive elements (TATC-box) in CsAAO6. We hypothesize that other plant AAOs within the same phylogenetic subgroup as CsAAO1 and CsAAO6 may similarly contain these specific cis-acting elements. Of particular interest, the LTR motif was detected in the promoter regions of seven CsAAOs (CsAAO1-2 and CsAAO4-8). Subsequently, the responses of the CsAAOs to cold stress were examined through qRT-PCR analysis (Figure 7). Remarkably, differential expression of the seven CsAAOs was observed at various time points following cold treatment, with CsAAO7 demonstrating the most pronounced upregulation (130.9-fold at 8 h). Unexpectedly, CsAAO7 expression was not detected in any of the eight tissues analyzed. Nevertheless, CsAAO7 expression was observed at very low levels in newly induced calluses and mature leaves (Supplementary Table S2), as well as in mesophyll protoplasts (our unpublished data). We hypothesize that CsAAO7 expression is regulated by cold treatment. Furthermore, AAO activity peaked after 12 h of exposure to low temperatures (Figure 8). Further investigation is warranted to elucidate the precise physiological function of this gene. In Ammopiptanthus nanus, AnAO5 was found to be involved in the low-temperature response, with low-temperature response cis-elements present in its promoter regions [16]. Subsequent functional analyses demonstrated that the overexpression of AnAO5 resulted in increased cold stress tolerance in Arabidopsis seedlings. These findings suggest a potentially important function of the AAO gene in the response of plants to cold stress, particularly in relation to low-temperature-response-related cis-elements.

5. Conclusions

Nine AAO genes were found in C. sinensis, with uneven distribution across four chromosomes. All CsAAOs have three conserved domains and are predicted to localize in the apoplast. The CsAAO gene family has diverse intron–exon patterns and is divided into three main clades. Three duplicate gene pairs were identified, evolving under purifying selection. Cis-acting elements in CsAAO promoters include hormone and stress response elements. Computational analysis suggests CsAAO9 may be targeted by csi-miR156. CsAAOs are highly expressed in roots and young fruits, with six CsAAOs upregulated in response to cold treatment. CsAAO activities initially decrease then increase after exposure to low temperatures. Our findings indicate that the CsAAO genes may play a role in the response to cold stress and could serve as potential target genes for breeding agricultural crops with enhanced cold resistance.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agriculture14091643/s1, Figure S1: Sequence logos of conserved motifs in CsAAOs, Table S1: Primers used in this study, Table S2: The expression patterns of CsAAO7 in the Citrus Pan-genome to Breeding Database.

Author Contributions

Methodology, X.X.; software, X.X. and L.W.; validation, X.M. and N.D.; resources, X.X.; data curation, X.X.; writing—original draft preparation, S.Z., M.L. and X.X.; writing—review and editing, X.X., L.J. and L.W.; supervision, X.X.; project administration, X.X. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (32372663, 32102326); the Key Research and Development Program of Jiangsu Province (BE2023328); and the Natural Science Foundation of Jiangsu Province (BK20230571).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study can be found in the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Chromosomal distribution of CsAAO gene family members. Pairs of segmentally duplicated genes are indicated by red lines.
Figure 1. Chromosomal distribution of CsAAO gene family members. Pairs of segmentally duplicated genes are indicated by red lines.
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Figure 2. Phylogenetic trees, conserved motifs, conserved domains, and gene structures of CsAAOs in the C. sinensis. (a) The phylogenetic tree was generated by MEGA-X software using the Neighbor-joining (NJ) method. (b) Six conserved motifs of CsAAO proteins. (c) Conserved motifs of CsAAO proteins. (d) Gene structures of CsAAOs.
Figure 2. Phylogenetic trees, conserved motifs, conserved domains, and gene structures of CsAAOs in the C. sinensis. (a) The phylogenetic tree was generated by MEGA-X software using the Neighbor-joining (NJ) method. (b) Six conserved motifs of CsAAO proteins. (c) Conserved motifs of CsAAO proteins. (d) Gene structures of CsAAOs.
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Figure 3. Phylogeny of the AAOs in C. sinensis, Oryza sativa, and Arabidopsis thaliana. The phylogenetic tree was generated utilizing the Neighbor-joining method with p-distance substitution model (gamma = 1) and 1000 bootstrap replicates in MEGA-X software.
Figure 3. Phylogeny of the AAOs in C. sinensis, Oryza sativa, and Arabidopsis thaliana. The phylogenetic tree was generated utilizing the Neighbor-joining method with p-distance substitution model (gamma = 1) and 1000 bootstrap replicates in MEGA-X software.
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Figure 4. Genome-wide synteny analysis for AAO genes among C. sinensis, Oryza sativa, and Arabidopsis thaliana.
Figure 4. Genome-wide synteny analysis for AAO genes among C. sinensis, Oryza sativa, and Arabidopsis thaliana.
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Figure 5. The distribution of Cis-acting elements in promoters of CsAAO genes.
Figure 5. The distribution of Cis-acting elements in promoters of CsAAO genes.
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Figure 6. The spatial expression patterns of the CsAAO genes.
Figure 6. The spatial expression patterns of the CsAAO genes.
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Figure 7. Expression profiles of the CsAAO genes in response to cold treatment. Different letters indicate significant differences (ANOVA analysis, p < 0.05).
Figure 7. Expression profiles of the CsAAO genes in response to cold treatment. Different letters indicate significant differences (ANOVA analysis, p < 0.05).
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Figure 8. Changes in CsAAO activity under low-temperature treatments. Different letters indicate significant differences (ANOVA analysis, p < 0.05).
Figure 8. Changes in CsAAO activity under low-temperature treatments. Different letters indicate significant differences (ANOVA analysis, p < 0.05).
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Table 1. Basic information for the CsAAO genes in the C. sinensis genome.
Table 1. Basic information for the CsAAO genes in the C. sinensis genome.
Gene NameGene IDAmino Acids LengthMolecular Weight
(kDa)		Isoelectric
Point (pI)		Instability
Index (II)		Grand Average of Hydropathicity (GRAVY)Subcellular
Localization
Predicted
CsAAO1Cs_ont_1g000870.158666.168.7840.14−0.266Extracell
CsAAO2Cs_ont_1g025500.153960.299.1734.78−0.199Extracell
CsAAO3Cs_ont_1g025510.154160.489.1539.82−0.164Extracell
CsAAO4Cs_ont_2g001690.154361.479.7343.86−0.259Extracell
CsAAO5Cs_ont_2g018990.153859.528.0938.34−0.227Extracell
CsAAO6Cs_ont_2g030090.158064.727.3732.56−0.318Extracell
CsAAO7Cs_ont_2g035780.155462.198.2732.50−0.252Extracell
CsAAO8Cs_ont_3g001440.154260.219.2730.76−0.227Extracell
CsAAO9Cs_ont_8g028600.154560.919.3237.26−0.234Extracell
Table 2. The Ka/Ks ratios for duplicate AAO genes in C. sinensis.
Table 2. The Ka/Ks ratios for duplicate AAO genes in C. sinensis.
Paralogous GenesKaKsKa/KsSelective Pressure
CsAAO2—CsAAO30.16891.66460.1015Purifying selection
CsAAO4—CsAAO50.14641.34490.1089Purifying selection
CsAAO8—CsAAO90.24401.49730.1629Purifying selection
Table 3. Prediction of microRNA-targeting CsAAO genes.
Table 3. Prediction of microRNA-targeting CsAAO genes.
Gene NamePredicted miRNA Target SitesmiRNA LengthExpectationInhibition
CsAAO9csi-miR156h213.5Cleavage
CsAAO9csi-miR156i213.5Cleavage
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Xu, X.; Miao, X.; Deng, N.; Liang, M.; Wang, L.; Jiang, L.; Zeng, S. Identification of Ascorbate Oxidase Genes and Their Response to Cold Stress in Citrus sinensis. Agriculture 2024, 14, 1643. https://doi.org/10.3390/agriculture14091643

AMA Style

Xu X, Miao X, Deng N, Liang M, Wang L, Jiang L, Zeng S. Identification of Ascorbate Oxidase Genes and Their Response to Cold Stress in Citrus sinensis. Agriculture. 2024; 14(9):1643. https://doi.org/10.3390/agriculture14091643

Chicago/Turabian Style

Xu, Xiaoyong, Xingchen Miao, Naiyi Deng, Mengge Liang, Lun Wang, Lijuan Jiang, and Shaohua Zeng. 2024. "Identification of Ascorbate Oxidase Genes and Their Response to Cold Stress in Citrus sinensis" Agriculture 14, no. 9: 1643. https://doi.org/10.3390/agriculture14091643

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