Next Article in Journal
MSA-ResNet: A Neural Network for Fine-Grained Instar Identification of Spodoptera frugiperda Larvae in Smart Agriculture
Previous Article in Journal
Quantitative Source Apportionment and Source-Oriented Health Risk of Heavy Metals in Soils: A Case Study of Yutian County in the Southern Margin of Tarim Basin, China
Previous Article in Special Issue
Free- and Bound-Form Terpenes in Sweet Potato Peel and Their Antifungal Activity Against Aspergillus flavus-Induced Tomato Spoilage
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 15
Issue 12
10.3390/agronomy15122722
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genome-Wide Identification, Characterization, and Expression Profiles of the CCO Gene Family in Raphanus sativus L. Response to Fusarium oxysporum Resistance

by
Yinbo Ma
1,†,
Wei Zuo
1,†,
Piaopiao Shi
1,
Xinyi Cao
1,
Yuchen Xia
1,
Run Tan
1,
Shuai Hao
1,
Hanyan Sun
1 and
Wenxing Hu
2,*
1
College of Horticulture and Landscape Architecture, Yangzhou University, Yangzhou 225009, China
2
College of Plant Protection, Yangzhou University, Yangzhou 225009, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(12), 2722; https://doi.org/10.3390/agronomy15122722
Submission received: 16 October 2025 / Revised: 19 November 2025 / Accepted: 24 November 2025 / Published: 26 November 2025
(This article belongs to the Special Issue Phytopathogens and Crop Diseases)
Browse Figures
Versions Notes

Abstract

Carotenoid cleavage oxygenases (CCOs) constitute a pivotal enzyme family that catalyze the oxidative cleavage of carotenoids to produce apocarotenoids, which are important signaling molecules involved in plant development, stress adaptation, and immunity responses. Notably, several CCOs, particularly NCED members, contribute to pathogen resistance by mediating abscisic acid (ABA) biosynthesis and apocarotenoid-derived defense signaling. While this gene family has been characterized in diverse plant species, its composition and functions in radish (Raphanus sativus L.) remain poorly understood. This study presents the comprehensive, genome-wide identification of the CCO gene family in radish, revealing 19 putative RsCCO members. A combination of phylogenetic, gene structure, chromosomal localization, and synteny analyses revealed conserved evolutionary patterns and species-specific diversification of the RsCCO family within the Brassicaceae. Promoter analysis identified an abundance of cis-acting regulatory elements related to phytohormone signaling, light perception, and stress responses, suggesting complex transcriptional regulation. Crucially, transcriptomic profiling following infection with Fusarium oxysporum revealed that two genes, RsNCED6 and RsNCED9b, were significantly upregulated in a resistant radish inbred line (‘T5’) compared to a susceptible line (‘T24’), identifying them as key candidate genes involved in ABA-mediated defense responses. This work provides a foundational characterization of the radish CCO gene family, offering novel insights into its role in carotenoid metabolism and pathogen defense.

1. Introduction

Carotenoids are a class of tetraterpenoid pigments widely distributed in plants, functioning as essential accessory pigments in photosynthesis for light harvesting and photoprotection. Beyond their photobiological roles, carotenoids serve as precursors for diverse signaling molecules that regulate plant growth, development, and responses to environmental stresses [1,2]. Among the most important carotenoid-derived compounds are the phytohormones abscisic acid (ABA) and strigolactones (SLs), which are integral to abiotic stress tolerance, pathogen defense, root architecture modulation, and interactions with symbiotic fungi and parasitic plants [3,4]. Thus, a comprehensive understanding of carotenoid biosynthesis and degradation is critical for elucidating plant metabolic networks and provides a theoretical basis for targeted crop improvement [5].
Carotenoid metabolism encompasses biosynthesis, storage, and catabolism, with catabolic processes primarily mediated by the CCO enzyme family [6]. CCOs are non-heme iron-dependent enzymes that catalyze the oxidative cleavage of specific double bonds in carotenoid backbones, producing a wide array of apocarotenoids [7]. These metabolites serve pivotal function in signal transduction, antioxidant activities, and disease defense [8,9]. Based on substrate specificity and sequence homology, plant CCOs are generally classified into two major subfamilies: 9-cis-epoxycarotenoid dioxygenases (NCEDs) and carotenoid cleavage dioxygenases (CCDs), the latter comprising CCD1, CCD4, CCD7, and CCD8 [10]. NCEDs specifically cleave 9-cis-epoxycarotenoids such as 9-cis-violaxanthin and 9-cis-neoxanthin to produce xanthoxin, the rate-limiting precursor in ABA biosynthesis [11,12]. Consequently, NCEDs are extensively implicated in plant responses to drought, salinity, and other abiotic stresses, as well as in pathogen defense via ABA-mediated signaling [13,14,15]. In contrast, the CCD4 subfamily preferentially cleaves cyclic carotenoids, and its products are often associated with tissue coloration and the synthesis of aromatic compounds [16]. Some CCD4 members are stress-inducible, suggesting additional roles in stress adaptation. CCD1 enzymes, with well-documented roles in volatile apocarotenoid production, typically act in the cytosol to generate aroma- and flavor-related compounds, while also contributing to stress-related signaling [17]. CCD7 and CCD8, on the other hand, have been extensively studied in strigolactone biosynthesis, functioning sequentially in plastids to catalyze key steps in the formation of this class of apocarotenoid-derived hormones [18]. Strigolactones are crucial regulators of shoot branching, root system architecture, and plant–microbe interactions, underscoring the diverse developmental and ecological functions of CCD7/8. Moreover, certain apocarotenoids, such as β-cyclocitral, act as oxidative stress signals that activate defense gene expression and enhance disease resistance [19,20].
The number, structure, and function of CCO gene family members vary across different plant species. For example, Arabidopsis thaliana possesses 9 CCO genes (including 5 NCEDs), whereas rapeseed (Brassica napus), which has undergone whole-genome polyploidization, contains up to 30 CCO members, indicating significant expansion and functional differentiation [13,21]. Functional studies in tomato and peanut have demonstrated that CCOs regulate fruit coloration, flavor compound biosynthesis, and pathogen defense. In tomato, CCO genes influence pigment accumulation and flavor-related metabolites, thereby influencing both fruit coloration and aroma profiles [22]. In peanuts, transcriptomic analyses under pathogen challenge indicate that CCO expression patterns may be associated with defense-related processes [23]. Importantly, several studies have revealed that CCOs are directly involved in pathogen resistance across diverse species. For instance, NCED genes are rapidly induced under fusarium infection in crops such as wheat and tomato, leading to enhanced abscisic acid (ABA) accumulation and activation of downstream defense pathways [24,25]. Moreover, apocarotenoid derivatives such as β-cyclocitral have been identified as signaling molecules that modulate plant resistance responses during plant–pathogen interactions [19,26]. Some apocarotenoids exhibit direct antimicrobial activity, whereas others function as signaling cues that activate plant immune responses, inducing pathogenesis-related (PR) genes and reinforcing cell wall defenses [27].
Radish (R. sativus), an important root vegetable crop in the Brassicaceae family, is cultivated worldwide for its nutrient-rich fleshy taproot [28]. However, its production is frequently threatened by soil-borne diseases, particularly fusarium wilt caused by F. oxysporum, which severely reduces yield and quality. Pathogen infection often triggers excessive accumulation of reactive oxygen species (ROS), leading to oxidative stress [29]. Given their roles in carotenoid catabolism and apocarotenoid-mediated signaling, CCOs may be key regulators in radish responses to such stresses [30]. Although the role of ABA in fusarium wilt resistance is well established, the specific contribution of the CCO gene family to this process remains poorly understood in most crops. Notably, the CCO gene family in radish has not yet been systematically characterized, and its potential role in defense against F. oxysporum is unknown.
The objectives of this study were: (1) to perform a genome-wide identification and characterization of the carotenoid cleavage oxygenase (CCO) gene family in radish; (2) to analyze their phylogenetic relationships, structural features, chromosomal distribution, and evolutionary patterns; and (3) to investigate the expression profiles of RsCCO genes in resistant and susceptible radish lines in response to F. oxysporum infection. This work provides a theoretical framework for advancing our understanding of carotenoid metabolism and disease resistance mechanisms in radish, while also offering valuable genetic resources for molecular breeding programs aimed at improving crop resilience.

2. Materials and Methods

2.1. Identification of CCO Gene Family Members

To comprehensively identify members of the CCO gene family in the radish genome [31], a combined method was performed using a combination of homology and domain-based validation. The Hidden Markov Model (HMM) profile for the CCO domain (PF03055) was obtained from the Pfam database (http://pfam.xfam.org/ accessed on 12 June 2025). This profile was used to screen the radish protein sequences with HMMER v3.3.2 software [21] using the hmmsearch command, applying an E-value cutoff of ≤1 × 10−5.
In parallel, known CCO protein sequences from A. thaliana were used as queries in BLASTP (version 2.17.0) searches against the radish protein database, with an E-value threshold of ≤1 × 10−10 and a minimum sequence identity of 70%. Candidate sequences from both searches were merged, and redundant entries were removed. The presence and completeness of the canonical CCO domain in all non-redundant candidates were verified using the SMART and Pfam databases. The same procedure was applied to identify CCO proteins in B. rapa and B. oleracea.

2.2. Phylogenetic and Gene Structure Analysis

To elucidate the evolutionary relationships of radish CCO genes, multiple sequence alignments of full-length CCO protein sequences from radish, A. thaliana, B. rapa, and B. oleracea were performed using ClustalW [5]. Subsequently, a Maximum Likelihood (ML) phylogenetic tree was constructed in MEGA 11 software [19], with branch support evaluated by 1000 bootstrap replicates. The optimal amino acid substitution model (JTT amino acid substitution model) was determined based on the Bayesian Information Criterion (BIC). Radish CCO genes were assigned to subfamilies based on clustering with orthologs from reference species.
Gene structure was analyzed by aligning each RsCCO coding sequence (CDS) with its corresponding genomic DNA sequence using the Gene Structure Display Server (GSDS; http://gsds.gao-lab.org/ accessed on 20 June 2025). Conserved motifs in RsCCD protein sequences were identified using MEME Suite v5.5.0 (https://meme-suite.org/ accessed on 20 June 2025), specifying a maximum of 10 motifs with widths of 6–50 amino acids. These analyses provided insights into structural diversity and functional conservation within the RsCCO family.

2.3. Chromosomal Localization and Synteny Analysis

The chromosomal positions of RsCCO genes were determined from the radish genome annotation file and visualized with MapChart2.2 [20]. To investigate gene family expansion, pairs of RsCCO genes located within a 100 kb region on the same chromosome were classified as tandem duplication events. To explore interspecific evolutionary relationships, whole-genome synteny analysis between radish and A. thaliana, B. rapa, and B. oleracea was performed using MCScanX [32] with default parameters. This analysis identified syntenic blocks and orthologous gene pairs. Syntenic blocks and orthologous gene pairs were identified, and the non-synonymous (Ka) to synonymous (Ks) substitution rate ratios (Ka/Ks) were calculated using KaKs_Calculator 3.0 [33] with the Nei–Gojobori (NG) method. A Ka/Ks ratio < 1 was interpreted as evidence of purifying selection following species divergence.

2.4. Promoter and Cis-Acting Element Analysis

To investigate potential transcriptional regulatory mechanisms, the 2000 bp region upstream of the transcription start site was extracted for each RsCCO gene. These promoter sequences were analyzed using the PlantCARE database [34] to predict cis-acting regulatory elements (CREs). The identified CREs were functionally categorized, and their distribution was visualized using TBtools (V2.376) software [35] to illustrate potential regulatory patterns.

2.5. Plant Growth and F. oxysporum Inoculations

Two radish inbred lines, ‘T5’ (highly resistant) and ‘T24’ (highly susceptible) to F. oxysporum, were used in this study. F. oxysporum strain was isolated from the farm at Yangzhou University, China. The pathogen was identified and verified based on morphological characteristics and further confirmed by sequencing of the internal transcribed spacer region. For inoculation experiments, the verified strain was propagated on potato dextrose agar plates for 7 days at 25 °C. Conidia were harvested by flooding the plates with sterile distilled water, gently scraping the colony surface, and filtering the suspension through sterile gauze to remove mycelial fragments. The spore concentration was adjusted to 3 × 106 conidia mL−1 using a hemocytometer.
Pathogen inoculation was performed using a modified root-dipping method [36]. Ten-day-old seedlings were grown at 25 °C under controlled conditions, roots were rinsed to remove soil, and plants were immersed in a spore suspension (3 × 106 conidia mL−1) for 30 min. Inoculated seedlings were transplanted into 50-well trays containing autoclaved soil and incubated at 25 °C and 80% relative humidity in darkness for 24 h, followed by growth under a 16 h light/8 h dark cycle at 25 °C and 60% relative humidity.
Disease symptoms were systematically monitored on a daily basis following inoculation. Susceptibility was assessed based on visible phenotypic changes, including wilting, chlorosis, and vascular tissue browning. In particular, phloem browning in the taproot was employed as a key diagnostic marker to track the progression of fusarium wilt. The severity of symptoms was quantitatively recorded to distinguish between the resistant inbred line (‘T5’) and the susceptible line (‘T24’). Root tissues were harvested at 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 days post-inoculation (DPI), with three biological replicates collected at each time point for symptom evaluation. Based on the observed phenotypic differences, representative time points (0, 1, 3, and 6 days) were subsequently selected for RT-qPCR analysis, thereby providing molecular evidence to support and validate the phenotypic observations.

2.6. Total RNA Extraction and Gene Expression Analysis

Total RNA was extracted from ‘T5’ and ‘T24’ plants using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). RNA quality and quantity were assessed via agarose gel electrophoresis and a Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Two micrograms of purified total RNA were reverse transcribed into cDNA using an oligo (dT) primer and TOPscript™ reverse transcriptase. Primers specific for all 19 identified RsCCO genes were designed using Primer 5.0 software (Supplementary Table S1). The quantitative real-time PCR (RT-qPCR) assay was performed using SYBR Green Supermix on a CFX96™ Real-Time System (Bio-Rad, Hercules, CA, USA) with the following thermal profile: 95 °C for 3 min, followed by 39 cycles of 95 °C for 15 s and 58 °C for 20 s. The radish actin gene (RsActin) served as an internal reference for normalization. Relative gene expression levels were calculated using the 2−∆∆Ct method.

3. Results

3.1. Identification and Physicochemical Properties of CCO Gene Family Members

To comprehensively profile the CCO gene family in radish, a genome-wide search was conducted utilizing sequence homology and domain-based strategies against publicly available radish genome assemblies. Utilizing HMM profiles specific to the RPE65 domain, in conjunction with BLASTP (version 2.17.0) homology searches, resulted in the identification of 19, 18, and 16 CCO family members in radish, cabbage, and Chinese cabbage, respectively. Each gene was assigned a unique designation based on its homology with Arabidopsis CCO genes. To characterize the encoded proteins, molecular weights (MW) were calculated and found to range from approximately 33,134.20 to 69,335.66 Da. Theoretical isoelectric points (pI) were noted to span from 4.89 to 9.88, indicating that the majority of RsaCCO proteins are weakly acidic, except for RsNCED2a and RsCCD8 (Table 1). Amino acid composition analysis, conducted using Expasy ProtParam and similar applications, revealed variability in the aliphatic index (indicative of thermodynamic stability) from 73.61 to 87.58. In addition, negative grand average of hydropathicity (GRAVY) values, ranging from –0.39 to 0.322, reflected the predominantly hydrophilic nature of these proteins. These physicochemical properties are highly consistent with those observed in CCO proteins from cabbage, Chinese cabbage, and Arabidopsis.

3.2. Phylogenetic Analysis and Classification of the CCO Family

To investigate the evolutionary relationships of the CCO gene family, phylogenetic analysis was performed using multiple CCO protein sequences from radish (19), Chinese cabbage (16), cabbage (18), and Arabidopsis (9). The phylogenetic tree robustly classified the 62 CCO proteins into four major subfamilies: CCD1, CCD4, CCD7/8, and NCED (Figure 1). Among these, the NCED subfamily was the largest, containing 32 members, whereas the CCD4 subfamily was the smallest, with 7 members. The CCD7 and CCD8 clustered together on one major branch, whereas the CCD1 and CCD4 subfamilies formed another distinct branch. All members of the NCED subfamily (NCED2, NCED3, NCED5, NCED6, and NCED9) were grouped tightly, indicating a close evolutionary kinship. Within each subfamily, CCO proteins from radish, cabbage, and Chinese cabbage generally formed sister clades, suggesting higher homology among these Brassica species compared to Arabidopsis. In contrast, the Arabidopsis CCOs appeared in separate clusters or at basal positions within branches, highlighting their greater evolutionary divergence. This topology aligns with known phylogenetic relationships and indicates a high degree of evolutionary conservation of the CCO gene family within the Brassica genus.

3.3. Gene Structure and Conserved Motif Analysis of the Radish CCO Gene Family

The exon-intron organization of the 19 RsCCO genes and the conserved motifs of their encoded proteins were analyzed to gain insight into their structural features and potential functions (Figure 2). A total of 10 conserved motifs were identified. RsCCO members within the same subfamily generally shared similar motif compositions and arrangements. For instance, all members of the RsCCD4 subfamily contained motifs 1, 2, 3, 5, 6, 8, and 10. Similarly, most members of the RsNCED subfamily displayed a highly consistent motif architecture. This strong conservation within subfamilies suggests that these genes may share common biological roles.
Gene structure analysis revealed significant variation in exon numbers and gene lengths. Members of the RsCCD1, RsCCD7, and RsCCD8 subfamilies possessed multiple exons and introns, resulting in relatively complex gene structures. In contrast, many NCED and CCD4 genes contained only a single exon, indicating simpler structure organization. When integrated with a phylogenetic tree, genes with similar gene structures and motif patterns clustered together, thereby reinforcing subfamily classifications. The observed diversity in exon-intron organization likely reflects evolutionary divergence, which may have contributed to functional specialization within the RsCCO family.

3.4. Chromosomal Localization and Synteny Analysis

The 19 RsCCO genes were mapped onto the radish genome to determine their physical locations and to explore potential duplication mechanisms. These genes were unevenly distributed across eight chromosomes (Figure 3). Chromosomes 1, 3, 8, and 9 each contained a single RsCCO gene, whereas chromosomes 2, 4, 5, and 6 harbored the majority. Two gene pairs, RsNCED2a/RsNCED2b and RsCCD1d/RsCCD1e, were identified as tandem duplicates, while 12 pairs were identified as segmental duplications. The Ka/Ks ratios of all duplicated pairs were less than one (Supplementary Table S2), indicating that purifying selection has constrained their evolutionary divergence.
This chromosomal distribution, together with the presence of both tandem and segmental duplications, suggests that the RsCCO family has expanded through multiple duplication mechanisms. Specifically, the coexistence of local tandem duplications and large-scale segmental rearrangements points to a dual contribution of small-scale and genome-wide processes in shaping the current gene repertoire. Moreover, the differential retention of duplicated genes across chromosomes implies that selective pressures have favored the preservation of functionally important paralogs while eliminating redundant copies. Such patterns of expansion and retention are consistent with the evolutionary trajectories observed in other Brassicaceae gene families, underscoring the central role of duplication-driven diversification in the structural and functional evolution of the RsCCO family.
Interspecific genomic synteny analysis revealed extensive collinearity between the RsCCO gene family and the CCO families of A. thaliana, B. rapa, and B. oleracea (Figure 4). A total of 27, 53, and 41 orthologous CCO gene pairs were identified between radish and A. thaliana, B. rapa, B. oleracea, respectively. The presence of these syntenic blocks provides strong evidence for tracing the orthologous relationships and evolutionary trajectories of CCO genes among these species, reflecting both conservation and divergence in genome structure and gene arrangement within the Brassicaceae family. Moreover, the observation that all Ka/Ks ratios were below 1 indicates that purifying selection has acted on these genes, eliminating most protein-altering mutations and thereby preserving their essential biological functions.

3.5. Prediction of Cis-Acting Elements in RsCCO Promoter Regions

To explore the transcriptional regulatory networks governing RsCCO genes, the 2000 bp upstream promoter regions of all 19 genes were analyzed for cis-acting regulatory elements using the PlantCARE database (Figure 5). A total of 39 distinct CREs were identified and classified into five major functional categories: (i) core promoter elements (e.g., TATA-box, CAAT-box); (ii) light-responsive elements (e.g., G-box, Sp1); (iii) hormone-responsive elements, including those related to abscisic acid (ABRE), auxin (AuxRR-core), gibberellin (GARE-motif), and salicylic acid (TCA-element); (iv) stress-responsive elements, such as MBS (drought), LTR (low temperature), and TC-rich repeats (defense); and (v) transcription factor binding sites (e.g., MYB, MYC). The abundance and diversity of these CREs, particularly those related to hormone signaling and stress responses, indicate that RsCCO gene expression is regulated by complex and multilayered networks. This intricate control likely enables their multifaceted roles in radish growth, development, and environmental adaptation, including defense against pathogens.

3.6. Expression Pattern Analysis of RsCCO Genes Under Fusarium Wilt Stress

To investigate the potential roles of the RsCCO family in biotic stress responses, phenotypic symptoms were compared between resistant and susceptible radish inbred lines following inoculation with F. oxysporum. Disease progression was closely monitored, revealing clear differences between the resistant line ‘T5’ and the susceptible line ‘T24’. At 6 DPI, ‘T24’ seedlings displayed clear signs of infection, including phloem browning, whereas ‘T5’ seedlings remained symptom-free (Figure 6). By 9 DPI, susceptible plants showed severe wilting and chlorosis. Based on these observations, early infection stages (0, 1, 3, and 6 DPI) were selected for subsequent gene expression analysis.
Of the 19 RsCCO genes, 7 genes (RsCCD1b/e, RsCCD7, RsNCED2a/b/c, and RsNCED9a) were not detectably expressed in root tissues under experimental conditions. Among the expressed genes, RsNCED6 and RsNCED9b showed striking resistance-specific upregulation (Figure 7). Transcript levels of both genes were consistently and significantly higher in the resistant ‘T5’ line compared with the susceptible ‘T24’ line across all post-inoculation time points. In contrast, the remaining expressed RsCCO genes exhibited no significant differences between the two lines. This targeted and robust upregulation in the resistant cultivar strongly implicates RsNCED6 and RsNCED9b as key candidate positive regulators in radish defense against F. oxysporum infection.

4. Discussion

CCOs are pivotal enzymes catalyzing the degradation of carotenoids into bioactive apocarotenoids, thereby playing crucial roles in plant growth, development, signal transduction, and environmental adaptation. Although the CCO gene family has been characterized in several model species, its systematic analysis and functional roles, and specific involvement in regulating disease resistance responses have not been thoroughly investigated in radish. This study provides comprehensive, genome-wide identification and characterization of the CCO gene family in radish. By integrating detailed phylogenetic, structural, and syntenic analyses with targeted gene expression profiling, we provide novel insights into the evolutionary dynamics of RsCCO genes and their potential roles in the defense response against F. oxysporum.

4.1. Genome-Wide Identification and Classification of the RsCCO Gene Family

The identification of 19 RsCCO genes represents a comprehensive characterization of this gene family in radish. These RsCCO genes were classified into four distinct subfamilies, corresponding to CCD1, CCD4, CCD7/8, and NCED, consistent with classifications A. thaliana, B. napus, and Nicotiana tabacum [37,38]. Subfamily assignments were strongly supported by conserved motif composition, exon–intron organization, and phylogenetic clustering, underscoring the high degree of structural and functional conservation among Brassicaceae orthologs.
All RsCCO proteins contained the RPE65 domain (PF03055), reflecting strong evolutionary constraints to preserve enzymatic core functions. Genes within the same subfamily exhibited greater similarity in motif arrangement and gene structure, suggesting subfamily-specific regulatory constraints and enzyme specialization have been preserved over evolutionary time [37,39]. Notably, most NCED genes in radish and other dicots are intronless, a feature thought to facilitate rapid transcriptional activation during stress, consistent with their established role in abscisic acid (ABA) biosynthesis [40].

4.2. Conserved Motifs, Gene Structure, and Implications for Enzymatic Function

The RsCCO family exhibits conserved motifs essential for carotenoid substrate recognition and catalysis, including four critical histidine residues required for iron binding and enzymatic activity. While CCD1 and CCD4 share several core motifs, the NCED and CCD7/8 subfamilies possess unique, subfamily-specific motifs, reflecting a divergence in substrate specificity and physiological function [37,38]. Phylogenetic analysis further supports this classification: the major subfamilies were resolved with bootstrap values exceeding 70%, indicating robust evolutionary grouping. In contrast, several internal nodes displayed lower bootstrap support, suggesting potential functional diversification among closely related members.
This motif landscape is remarkably congruent with the observed gene structures. NCED genes are typically intronless (single-exon), a feature associated with rapid inducibility, whereas CCD1/4 genes possess multiple exons, suggesting greater regulatory complexity and functional diversification. The analysis of exon-intron architecture revealed both conservation and divergence within the gene family. The intron-free architecture of NCEDs, widespread among higher plants, is hypothesized to confer a selective advantage by enabling the rapid upregulation of ABA biosynthesis in response to sudden abiotic and biotic stressors [41]. Conversely, the more intricate exon–intron structures found in the CCD1/4 and CCD7/8 subfamilies may reflect their involvement in developmentally programmed or tissue-specific carotenoid cleavage, leading to the formation of specialized apocarotenoids and strigolactones—metabolites known to govern plant architecture, pigmentation, and root–microbe communication [42,43].

4.3. Chromosomal Distribution, Synteny, and Evolutionary Dynamics in Brassicaceae

Chromosomal mapping revealed an uneven distribution of RsCCO genes, a pattern likely shaped by genome duplication and segmental gene loss following the ancestral whole-genome triplication (WGT) event ancestral to the Brassicaceae [28,44]. The presence of RsCCO gene clusters and collinearity with orthologous regions in other Brassica species further corroborates the profound influence of WGT on the gene family expansion and diversification. Importantly, Ks and Ka/Ks analyses consistently demonstrated that purifying selection is the dominant evolutionary pressure acting on these genes. This finding aligns with observations across plant taxa and highlights the critical importance of maintaining CCO gene functionality for plant survival [26,45]. Furthermore, Ks distribution analyses and syntenic comparisons indicate that the duplication events underpinning the current complement of RsCCO genes are ancient, predating speciation within Brassicaceae and closely paralleling the estimated timeline of the WGT event (~15–25 Mya). These synapomorphic events, combined with segmental rearrangements and selection for functionally important paralogs, are pivotal to the emergence of lineage- and tissue-specific gene regulatory networks.

4.4. Functional Roles of the CCO Family: Integrating ABA and Plant Defense

A well-established role for the CCO gene family across the plant kingdom, especially for members of the NCED subfamily, is its central involvement in the biosynthesis of the key plant hormones ABA and strigolactones (SLs), which are crucial for both development and defense responses [39,43,46]. NCED-mediated cleavage of 9-cis-epoxycarotenoids is the rate-limiting step in ABA synthesis, a mechanism confirmed in both model and crop species [37,38]. For instance, functional analyses of Arabidopsis orthologs, AtNCED6 and AtNCED9, reveal tissue-specific expression patterns and key regulatory roles in seed development, dormancy, and stress response [40]. In radish, gene expression profiling under F. oxysporum infection revealed pronounced upregulation of RsNCED6 and RsNCED9b in resistant lines, implicating them as candidate regulators of defense. Although ABA levels and antioxidant enzyme activities (POD, SOD, PAL) were not directly measured, the transcriptional induction of RsNCED genes suggests enhanced ABA biosynthesis, consistent with reports linking ABA to increased antioxidant activity and reduced lipid peroxidation [47,48].
These findings are congruent with broader evidence suggesting a positive correlation between ABA-mediated signaling and enhanced resistance to F. oxysporum, particularly during the early phases of infection. In this context, ABA can directly or indirectly regulate the expression of pathogenesis-related genes, promote cell wall modifications, and modulate cellular redox status [47,49]. It is important to note that while ABA often exhibits an antagonistic relationship with salicylic acid (SA)–mediated responses to certain pathogens, its role can be positive in the context of vascular wilt pathogens like F. oxysporum. This is likely achieved by promoting rapid stomatal closure, orchestrating callose deposition, and enhancing antioxidant defenses [43,49].

4.5. Limitations and Future Perspectives

This study provides a foundational, genome-wide analysis of the RsCCO family and presents compelling evidence for its role in disease resistance. However, these findings are primarily based on bioinformatic predictions and transcript-level analysis, which have inherent limitations. Future research must focus on the experimental validation of these putative functions. A critical priority is the quantification of endogenous ABA levels in resistant (‘T5’) and susceptible (‘T24’) radish roots following F. oxysporum infection, to determine whether transcriptional induction of RsNCED6 and RsNCED9b translates into enhanced ABA biosynthesis. In parallel, functional studies using CRISPR/Cas9-mediated gene editing or virus-induced gene silencing (VIGS) to generate transgenic lines with targeted overexpression or silencing of these genes will enable direct assessment of their causal roles in fusarium wilt resistance. Integrating ABA measurements with phenotypic and molecular analyses will provide decisive evidence linking RsNCED activity to ABA-mediated defense pathways and support breeding strategies for improved crop resilience.

5. Conclusions

This study provides a systematic characterization of the CCO gene family in R. sativus. Our analysis of their structural features, phylogenetic relationships, and transcriptional responses to fusarium wilt infection elucidates the multifaceted roles of these genes in stress resilience, hormone biosynthesis, and plant development. Our findings highlight RsNCED6 and RsNCED9b as high-priority candidates for future functional genomics studies and for potential application in crop improvement programs. The regulatory complexity suggested by their cis-acting elements underscores the potential for sophisticated genetic and biotechnological interventions to bolster disease resistance in radish. Addressing the current limitations through rigorous functional validation will be critical to realizing this potential, thereby providing a robust foundation for both fundamental and applied advances in plant pathology and crop breeding.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15122722/s1, Table S1: Forward and reverse primer sequences of RsCCO genes; Table S2: ka/ks value of RsCCO in radish.

Author Contributions

Conceptualization and manuscript revision: W.H.; methodology: Y.M. and W.Z.; data analysis and draft writing: Y.M. and W.Z.; experiments and figure and table preparation: W.Z., P.S., X.C., Y.X. and R.T.; manuscript review and improvement: W.H., S.H. and H.S. All authors reviewed the results. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from the Natural Science Foundation of Jiangsu Province (BK20220573 and BK20240904).

Data Availability Statement

The original contributions of this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Nisar, N.; Li, L.; Lu, S.; Khin, N.; Pogson, B. Carotenoid metabolism in plants. Mol. Plant 2015, 8, 68–82. [Google Scholar] [CrossRef]
  2. Yuan, H.; Zhang, J.; Nageswaran, D.; Li, L. Carotenoid metabolism and regulation in horticultural crops. Hortic. Res. 2015, 2, 15036. [Google Scholar] [CrossRef]
  3. Al-Babili, S.; Bouwmeester, H. Strigolactones, a novel carotenoid-derived plant hormone. Annu. Rev. Plant Biol. 2015, 66, 161–186. [Google Scholar] [CrossRef] [PubMed]
  4. Jia, K.-P.; Li, C.; Bouwmeester, H.J.; Al-Babili, S. Strigolactone biosynthesis and signal transduction. In Strigolactones-Biology and Applications; Springer International Publishing: Cham, Switzerland, 2019; pp. 1–45. [Google Scholar]
  5. Rella, A.; Farnoud, A.M.; Del Poeta, M. Plasma membrane lipids and their role in fungal virulence. Prog. Lipid Res. 2016, 61, 63–72. [Google Scholar] [CrossRef]
  6. Auldridge, M.E.; McCarty, D.R.; Klee, H.J. Plant carotenoid cleavage oxygenases and their apocarotenoid products. Curr. Opin. Plant Biol. 2006, 9, 315–321. [Google Scholar] [CrossRef] [PubMed]
  7. Harrison, P.J.; Bugg, T.D. Enzymology of the carotenoid cleavage dioxygenases: Reaction mechanisms, inhibition and biochemical roles. Arch. Biochem. Biophys. 2014, 544, 105–111. [Google Scholar] [CrossRef] [PubMed]
  8. Havaux, M. Carotenoid oxidation products as stress signals in plants. Plant J. 2014, 79, 597–606. [Google Scholar] [CrossRef]
  9. Walter, M.H.; Floss, D.S.; Strack, D. Apocarotenoids: Hormones, mycorrhizal metabolites and aroma volatiles. Planta 2010, 232, 1–17. [Google Scholar] [CrossRef]
  10. Heier, C.; Haemmerle, G. Fat in the heart: The enzymatic machinery regulating cardiac triacylglycerol metabolism. Biochim. Biophys. Acta (BBA)-Mol. Cell Biol. Lipids 2016, 1861, 1500–1512. [Google Scholar] [CrossRef]
  11. Schwartz, S.H.; Tan, B.C.; Gage, D.A.; Zeevaart, J.A.; McCarty, D.R. Specific oxidative cleavage of carotenoids by VP14 of maize. Science 1997, 276, 1872–1874. [Google Scholar] [CrossRef]
  12. Tan, B.C.; Schwartz, S.H.; Zeevaart, J.A.; McCarty, D.R. Genetic control of abscisic acid biosynthesis in maize. Proc. Natl. Acad. Sci. USA 1997, 94, 12235–12240. [Google Scholar] [CrossRef]
  13. Iuchi, S.; Kobayashi, M.; Taji, T.; Naramoto, M.; Seki, M.; Kato, T.; Tabata, S.; Kakubari, Y.; Yamaguchi-Shinozaki, K.; Shinozaki, K. Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J. 2001, 27, 325–333. [Google Scholar] [CrossRef]
  14. Nambara, E.; Marion-Poll, A. Abscisic acid biosynthesis and catabolism. Annu. Rev. Plant Biol. 2005, 56, 165–185. [Google Scholar] [CrossRef] [PubMed]
  15. Li, M.; Yu, G.; Cao, C.; Liu, P. Metabolism, signaling, and transport of jasmonates. Plant Commun. 2021, 2, 100231. [Google Scholar] [CrossRef] [PubMed]
  16. Ohmiya, A.; Kishimoto, S.; Aida, R.; Yoshioka, S.; Sumitomo, K. Carotenoid cleavage dioxygenase (CmCCD4a) contributes to white color formation in chrysanthemum petals. Plant Physiol. 2006, 142, 1193–1201. [Google Scholar] [CrossRef] [PubMed]
  17. Floss, D.S.; Walter, M.H. Role of carotenoid cleavage dioxygenase 1 (CCD1) in apocarotenoid biogenesis revisited. Plant Signal. Behav. 2009, 4, 172–175. [Google Scholar] [CrossRef]
  18. Bruno, M.; Koschmieder, J.; Wuest, F.; Schaub, P.; Fehling-Kaschek, M.; Timmer, J.; Beyer, P.; Al-Babili, S. Enzymatic study on AtCCD4 and AtCCD7 and their potential to form acyclic regulatory metabolites. J. Exp. Bot. 2016, 67, 5993–6005. [Google Scholar] [CrossRef]
  19. Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef]
  20. Voorrips, R. MapChart: Software for the graphical presentation of linkage maps and QTLs. J. Hered. 2002, 93, 77–78. [Google Scholar] [CrossRef]
  21. Eddy, S.R. Accelerated profile HMM searches. PLoS Comput. Biol. 2011, 7, e1002195. [Google Scholar] [CrossRef]
  22. Simkin, A.J.; Schwartz, S.H.; Auldridge, M.; Taylor, M.G.; Klee, H.J. The tomato carotenoid cleavage dioxygenase 1 genes contribute to the formation of the flavor volatiles β-ionone, pseudoionone, and geranylacetone. Plant J. 2004, 40, 882–892. [Google Scholar] [CrossRef]
  23. Liu, H.; Micic, N.; Miller, S.; Crocoll, C.; Bjarnholt, N. Species-specific dynamics of specialized metabolism in germinating sorghum grain revealed by temporal and tissue-resolved transcriptomics and metabolomics. Plant Physiol. Biochem. 2023, 196, 807–882. [Google Scholar] [CrossRef]
  24. Su, W.; Zhang, C.; Feng, J.; Feng, A.; You, C.; Ren, Y.; Wang, D.; Sun, T.; Su, Y.; Xu, L. Genome-wide identification, characterization and expression analysis of the carotenoid cleavage oxygenase (CCO) gene family in Saccharum. Plant Physiol. Biochem. 2021, 162, 196–210. [Google Scholar] [CrossRef]
  25. Hao, G.; Usgaard, T.; Tiley, H.; McCormick, S.; Vaughan, M.; Ward, T. Trichothecene NX-3 is required for Fusarium graminearum initial infection and disease spread. In Meeting Abstract; National Center for Agricultural Utilization Research: Peoria, IL, USA, 2021. [Google Scholar]
  26. Singh, N.K.; Anand, S.; Jain, A.; Das, S. Comparative genomics and synteny analysis of KCS17-KCS18 cluster across different genomes and sub-genomes of brassicaceae for analysis of its evolutionary history. Plant Mol. Biol. Report. 2017, 35, 237–251. [Google Scholar] [CrossRef]
  27. Sun, G.; Liao, J.; Kurze, E.; Hoffmann, T.D.; Steinchen, W.; McGraphery, K.; Habegger, R.; Marek, L.; Catici, D.A.; Ludwig, C. Apocarotenoids are allosteric effectors of a dimeric plant glycosyltransferase involved in defense and lignin formation. New Phytol. 2023, 238, 2080–2098. [Google Scholar] [CrossRef] [PubMed]
  28. Mitsui, Y.; Shimomura, M.; Komatsu, K.; Namiki, N.; Shibata-Hatta, M.; Imai, M.; Katayose, Y.; Mukai, Y.; Kanamori, H.; Kurita, K. The radish genome and comprehensive gene expression profile of tuberous root formation and development. Sci. Rep. 2015, 5, 1083. [Google Scholar] [CrossRef]
  29. Torres, M.A.; Jones, J.D.; Dangl, J.L. Reactive oxygen species signaling in response to pathogens. Plant Physiol. 2006, 141, 373–378. [Google Scholar] [CrossRef] [PubMed]
  30. Walter, M.H.; Strack, D. Carotenoids and their cleavage products: Biosynthesis and functions. Nat. Prod. Rep. 2011, 28, 663–692. [Google Scholar] [CrossRef] [PubMed]
  31. Xu, L.; Wang, Y.; Dong, J.; Zhang, W.; Tang, M.; Zhang, W.; Wang, K.; Chen, Y.; Zhang, X.; He, Q. A chromosome-level genome assembly of radish (Raphanus sativus L.) reveals insights into genome adaptation and differential bolting regulation. Plant Biotechnol. J. 2023, 21, 990–1004. [Google Scholar] [CrossRef]
  32. Wang, Y.; Tang, H.; DeBarry, J.D.; Tan, X.; Li, J.; Wang, X.; Lee, T.-H.; Jin, H.; Marler, B.; Guo, H. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012, 40, e49. [Google Scholar] [CrossRef]
  33. Zhang, Z. KaKs_Calculator 3.0: Calculating selective pressure on coding and non-coding sequences. Genom. Proteom. Bioinform. 2022, 20, 536–540. [Google Scholar] [CrossRef]
  34. Lescot, M.; Déhais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouzé, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325–327. [Google Scholar] [CrossRef] [PubMed]
  35. Chen, C.; Wu, Y.; Li, J.; Wang, X.; Zeng, Z.; Xu, J.; Liu, Y.; Feng, J.; Chen, H.; He, Y. TBtools-II: A “one for all, all for one” bioinformatics platform for biological big-data mining. Mol. Plant 2023, 16, 1733–1742. [Google Scholar] [CrossRef] [PubMed]
  36. Yu, X.; Choi, S.R.; Ramchiary, N.; Miao, X.; Lee, S.H.; Sun, H.J.; Kim, S.; Ahn, C.H.; Lim, Y.P. Comparative mapping of Raphanus sativus genome using Brassica markers and quantitative trait loci analysis for the Fusarium wilt resistance trait. Theor. Appl. Genet. 2013, 126, 2553–2562. [Google Scholar] [CrossRef]
  37. Zhou, X.-T.; Jia, L.-D.; Duan, M.-Z.; Chen, X.; Qiao, C.-L.; Ma, J.-Q.; Zhang, C.; Jing, F.-Y.; Zhang, S.-S.; Yang, B. Genome-wide identification and expression profiling of the carotenoid cleavage dioxygenase (CCD) gene family in Brassica napus L. PLoS ONE 2020, 15, e0238179. PLoS ONE 2020, 15, e0238179. [Google Scholar]
  38. Zhou, Q.; Li, Q.; Li, P.; Zhang, S.; Liu, C.; Jin, J.; Cao, P.; Yang, Y. Carotenoid cleavage dioxygenases: Identification, expression, and evolutionary analysis of this gene family in tobacco. Int. J. Mol. Sci. 2019, 20, 5796. [Google Scholar] [CrossRef] [PubMed]
  39. Li, H.; Yao, X.; He, A.; Xue, G.; Yang, H.; Fan, Y.; Yang, S.; Ruan, J. Genome-wide identification and gene expression pattern analysis of the carotenoid cleavage oxygenase gene family in Fagopyrum tataricum. BMC Plant Biol. 2025, 25, 466. [Google Scholar] [CrossRef]
  40. Lefebvre, V.; North, H.; Frey, A.; Sotta, B.; Seo, M.; Okamoto, M.; Nambara, E.; Marion-Poll, A. Functional analysis of Arabidopsis NCED6 and NCED9 genes indicates that ABA synthesized in the endosperm is involved in the induction of seed dormancy. Plant J. 2006, 45, 309–319. [Google Scholar] [CrossRef]
  41. Sato, H.; Takasaki, H.; Takahashi, F.; Suzuki, T.; Iuchi, S.; Mitsuda, N.; Ohme-Takagi, M.; Ikeda, M.; Seo, M.; Yamaguchi-Shinozaki, K. Arabidopsis thaliana NGATHA1 transcription factor induces ABA biosynthesis by activating NCED3 gene during dehydration stress. Proc. Natl. Acad. Sci. USA 2018, 115, E11178–E11187. [Google Scholar] [CrossRef]
  42. Cheng, C.; Yang, R.; Yin, L.; Zhang, J.; Gao, L.; Lu, R.; Yang, Y.; Wang, P.; Mu, X.; Zhang, S. Characterization of carotenoid cleavage oxygenase genes in Cerasus humilis and functional analysis of ChCCD1. Plants 2023, 12, 2114. [Google Scholar] [CrossRef]
  43. Zheng, X.; Yang, Y.; Al-Babili, S. Exploring the diversity and regulation of apocarotenoid metabolic pathways in plants. Front. Plant Sci. 2021, 12, 787049. [Google Scholar] [CrossRef]
  44. Jeong, Y.-M.; Chung, W.-H.; Chung, H.; Kim, N.; Park, B.-S.; Lim, K.-B.; Yu, H.-J.; Mun, J.-H. Comparative analysis of the radish genome based on a conserved ortholog set (COS) of Brassica. Theor. Appl. Genet. 2014, 127, 1975–1989. [Google Scholar] [CrossRef]
  45. Walden, N.; Schranz, M.E. Synteny identifies reliable orthologs for phylogenomics and comparative genomics of the Brassicaceae. Genome Biol. Evol. 2023, 15, evad034. [Google Scholar] [CrossRef]
  46. Zeng, L.; Zeng, L.; Wang, Y.; Xie, Z.; Zhao, M.; Chen, J.; Ye, X.; Tie, W.; Li, M.; Shang, S. Identification and expression of the CCO family during development, ripening and stress response in banana. Genetica 2023, 151, 87–96. [Google Scholar] [CrossRef] [PubMed]
  47. Boba, A.; Kostyn, K.; Kozak, B.; Wojtasik, W.; Preisner, M.; Prescha, A.; Gola, E.M.; Lysh, D.; Dudek, B.; Szopa, J. Fusarium oxysporum infection activates the plastidial branch of the terpenoid biosynthesis pathway in flax, leading to increased ABA synthesis. Planta 2020, 251, 50. [Google Scholar] [CrossRef] [PubMed]
  48. He, R.; Chang, Y.; Wang, J. Identification of genes responsible for stress resistance in Fusarium oxysporum-inoculated flax seedlings using weighted gene co-expression network analysis. Eur. J. Plant Pathol. 2022, 163, 513–528. [Google Scholar] [CrossRef]
  49. Kaushal, M.; Mahuku, G.; Swennen, R. Comparative transcriptome and expression profiling of resistant and susceptible banana cultivars during infection by Fusarium oxysporum. Int. J. Mol. Sci. 2021, 22, 3002. [Google Scholar] [CrossRef]
Agronomy 15 02722 g001
Figure 1. Phylogenesis of the CCD Family in Radish, Chinese Cabbage, Cabbage, and Arabidopsis. The dendrogram was drawn by IQTree with the maximum likelihood method (ML) and JTT amino acid substitution model.
Figure 1. Phylogenesis of the CCD Family in Radish, Chinese Cabbage, Cabbage, and Arabidopsis. The dendrogram was drawn by IQTree with the maximum likelihood method (ML) and JTT amino acid substitution model.
Agronomy 15 02722 g001
Agronomy 15 02722 g002
Figure 2. Phylogenetic relationships, conserved motifs, and gene structure of CCO genes in radish. (a) The phylogenetic tree was constructed using the maximum likelihood method (ML) (JTT amino acid substitution model). Sub-families are distinguished by different colors; (b) the conserved motifs of the RsCCO proteins were identified by MEME; (c) exon/intron architectures of RsCCO genes in radish. Green and red boxes indicate exons and UTRs, respectively. Black lines represent introns. The size of exons and introns can be calculated according to the scale at the bottom.
Figure 2. Phylogenetic relationships, conserved motifs, and gene structure of CCO genes in radish. (a) The phylogenetic tree was constructed using the maximum likelihood method (ML) (JTT amino acid substitution model). Sub-families are distinguished by different colors; (b) the conserved motifs of the RsCCO proteins were identified by MEME; (c) exon/intron architectures of RsCCO genes in radish. Green and red boxes indicate exons and UTRs, respectively. Black lines represent introns. The size of exons and introns can be calculated according to the scale at the bottom.
Agronomy 15 02722 g002
Agronomy 15 02722 g003
Figure 3. Chromosomal localization and collinearity analysis of the RsCCO gene in radish chromosome. (a) Distribution of the RsCCO gene in radish chromosomes. Red boxes indicate pairs of tandem duplication events. (b) Collinearity analysis of the RsCCO gene in radish. Red lines indicate duplicated gene pairs.
Figure 3. Chromosomal localization and collinearity analysis of the RsCCO gene in radish chromosome. (a) Distribution of the RsCCO gene in radish chromosomes. Red boxes indicate pairs of tandem duplication events. (b) Collinearity analysis of the RsCCO gene in radish. Red lines indicate duplicated gene pairs.
Agronomy 15 02722 g003
Agronomy 15 02722 g004
Figure 4. Homology analysis of CCO genes among radish, Cabbage, Chinese Cabbage, and A. thaliana. Red lines indicate duplicated gene pairs, whereas gray lines represent all syntenic blocks in the radish genome.
Figure 4. Homology analysis of CCO genes among radish, Cabbage, Chinese Cabbage, and A. thaliana. Red lines indicate duplicated gene pairs, whereas gray lines represent all syntenic blocks in the radish genome.
Agronomy 15 02722 g004
Agronomy 15 02722 g005
Figure 5. Analysis of cis-regulatory elements in the promoter region of the RsCCO gene. (a) The phylogenetic tree was constructed using the maximum likelihood method (ML); (b) the distribution of cis-regulatory elements on the RsCCO gene promoter.
Figure 5. Analysis of cis-regulatory elements in the promoter region of the RsCCO gene. (a) The phylogenetic tree was constructed using the maximum likelihood method (ML); (b) the distribution of cis-regulatory elements on the RsCCO gene promoter.
Agronomy 15 02722 g005
Agronomy 15 02722 g006
Figure 6. Symptoms of radish after inoculation with F. oxysporum. (a) Growth status of resistant line ‘T5’ recorded daily after inoculation; (b) growth status of resistant line ‘T24’ recorded daily after inoculation; browning of the vascular bundle is indicated by the red arrow.
Figure 6. Symptoms of radish after inoculation with F. oxysporum. (a) Growth status of resistant line ‘T5’ recorded daily after inoculation; (b) growth status of resistant line ‘T24’ recorded daily after inoculation; browning of the vascular bundle is indicated by the red arrow.
Agronomy 15 02722 g006
Agronomy 15 02722 g007
Figure 7. Relative expression of RsCCO genes in response to F. oxysporum infection in radish. Asterisks indicate a significant difference between the ‘T5’ (highly resistant) and ‘T24’ (highly susceptible) for each gene, as determined by a two-tailed Student’s t-test (* p < 0.05, ** p < 0.01).
Figure 7. Relative expression of RsCCO genes in response to F. oxysporum infection in radish. Asterisks indicate a significant difference between the ‘T5’ (highly resistant) and ‘T24’ (highly susceptible) for each gene, as determined by a two-tailed Student’s t-test (* p < 0.05, ** p < 0.01).
Agronomy 15 02722 g007
Table 1. Physicochemical properties of RsCCO family members in R. sativus.
Table 1. Physicochemical properties of RsCCO family members in R. sativus.
Sequence IDRenameGene PositionAmino NO.CDS LengthMolecular WeightTheoretical pIInstability IndexAliphatic Index
Rsa1g020070.1RsNCED5Rs11143211111434169589177065,319.915.5944.4581.09
Rsa2g001310.1RsNCED9aRs2874445876160571171663,884.736.0042.7080.49
Rsa2g034050.1RsNCED2aRs2499013794990207723269925,757.879.8849.2887.07
Rsa2g034060.1RsNCED2bRs24990209149903110339102038,160.354.8943.3087.58
Rsa2g034800.1RsCCD4aRs25030650350308552596179165,701.836.3446.7080.79
Rsa2g044970.1RsCCD8Rs25582395955827221567170463,959.947.1432.1280.95
Rsa3g018990.1RsCCD1aRs3318012203180166312337213,990.176.0047.0776.75
Rsa4g010680.1RsNCED2cRs463063546308105583175264,952.985.3847.5086.28
Rsa4g035880.1RsCCD1bRs444228908442294889528810,682.195.4122.8483.05
Rsa4g050050.1RsCCD7Rs45418044754183265617185469,335.666.0145.8973.61
Rsa4g059070.1RsCCD1cRs46204550062049018534160560,191.095.6735.3483.18
Rsa5g009680.1RsNCED3aRs560083736010600597179465,683.135.6842.6778.56
Rsa5g017550.1RsNCED6Rs51215704012158797585175865,152.365.7239.1782.91
Rsa5g051130.1RsNCED3bRs54681983946821617592177965,437.045.7044.9078.72
Rsa6g006820.1RsNCED3cRs637219153724016598179765,802.375.9044.2776.62
Rsa6g044170.1RsCCD1dRs6498134374981733525677129,094.446.5035.2775.00
Rsa6g044210.1RsCCD1eRs64987338849876648432129949,328.986.2930.5783.03
Rsa8g021180.1RsCCD4bRs81261220412614151578173763,266.256.7242.7687.35
Rsa9g001690.1RsNCED9bRs912447821246776610183367,759.825.9749.3479.54
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Ma, Y.; Zuo, W.; Shi, P.; Cao, X.; Xia, Y.; Tan, R.; Hao, S.; Sun, H.; Hu, W. Genome-Wide Identification, Characterization, and Expression Profiles of the CCO Gene Family in Raphanus sativus L. Response to Fusarium oxysporum Resistance. Agronomy 2025, 15, 2722. https://doi.org/10.3390/agronomy15122722

AMA Style

Ma Y, Zuo W, Shi P, Cao X, Xia Y, Tan R, Hao S, Sun H, Hu W. Genome-Wide Identification, Characterization, and Expression Profiles of the CCO Gene Family in Raphanus sativus L. Response to Fusarium oxysporum Resistance. Agronomy. 2025; 15(12):2722. https://doi.org/10.3390/agronomy15122722

Chicago/Turabian Style

Ma, Yinbo, Wei Zuo, Piaopiao Shi, Xinyi Cao, Yuchen Xia, Run Tan, Shuai Hao, Hanyan Sun, and Wenxing Hu. 2025. "Genome-Wide Identification, Characterization, and Expression Profiles of the CCO Gene Family in Raphanus sativus L. Response to Fusarium oxysporum Resistance" Agronomy 15, no. 12: 2722. https://doi.org/10.3390/agronomy15122722

APA Style

Ma, Y., Zuo, W., Shi, P., Cao, X., Xia, Y., Tan, R., Hao, S., Sun, H., & Hu, W. (2025). Genome-Wide Identification, Characterization, and Expression Profiles of the CCO Gene Family in Raphanus sativus L. Response to Fusarium oxysporum Resistance. Agronomy, 15(12), 2722. https://doi.org/10.3390/agronomy15122722

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop