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Article

Expression Regulatory Mechanisms of the Key Structural Genes in the Carotenoid Biosynthesis Pathway Under Salt Stress of Lycium barbarum

by
Zhi-Hang Hu
1,
Li-Xiang Wang
1,2,
Nan Zhang
1,
Chen Chen
1,
Jing Zhuang
1,
Yue Yin
3,* and
Ai-Sheng Xiong
1,2,*
1
State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, Ministry of Agriculture and Rural Affairs Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
2
Suqian Research Institute of Nanjing Agricultural University, Facility Horticulture Research Institute of Suqian, Suqian 223800, China
3
National Wolfberry Engineering Research Center, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan 751002, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(10), 1149; https://doi.org/10.3390/horticulturae11101149
Submission received: 1 August 2025 / Revised: 11 September 2025 / Accepted: 17 September 2025 / Published: 24 September 2025
(This article belongs to the Special Issue New Insights into Protected Horticulture Stress)
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Abstract

Salt stress is a major abiotic factor limiting wolfberry (Lycium barbarum) growth. As a high-value medicinal and edible crop, wolfberry relies on its carotenoid content, a critical determinant of fruit quality and nutritional value. To elucidate the expression regulatory mechanisms of key genes in the carotenoid biosynthesis pathway under salt stress, this study systematically identified 17 structural genes within the L. barbarum carotenoid pathway using genomic and transcriptomic approaches. Comprehensive analyses were conducted on gene structure, chromosomal distribution, conserved domains, and cis-acting elements. The results revealed that these genes were clustered on chromosomes Chr08 and Chr10 and exhibit strong collinearity with tomato (18 syntenic pairs). Their promoters were enriched with light-responsive (G-box) and stress-responsive (ABRE, DRE) elements. Tissue-specific expression analysis demonstrated high expression in mid-to-late fruit developmental stages (LbaPSY1, LbaPDS) and in photoprotective genes (LbaZEP, LbaVDE) in leaves. Under 300 mM NaCl stress treatment, the genes exhibited a staged response: Early stage (1–3 h): upstream MEP pathway genes (LbaDXS, LbaGGPS) were rapidly induced to supply precursors. Mid-stage (6–12 h): midstream genes (LbaPSY, LbaPDS, LbaZDS) were continuously upregulated, promoting lycopene synthesis and preferentially activating the β-branch (LbaLCYB). Late stage (12–24 h): downstream xanthophyll cycle genes (LbaBCH, LbaZEP, LbaVDE) were significantly enhanced, facilitating the accumulation of antioxidant compounds like violaxanthin and neoxanthin. This coordinated regulation formed a synergistic “precursor supply–antioxidant product” network. This study revealed the phased and coordinated regulatory network of carotenoid biosynthesis genes under salt stress in L. barbarum. It also provided potential target genes for the new cultivar selection with enhanced salt tolerance and nutritional quality.

1. Introduction

Lycium barbarum L. (wolfberry), a high-value economic crop in the Solanaceae family and a strategic “medicine-food homology” resource, has attracted considerable research attention due to its core bioactive component—carotenoids, whose biosynthesis regulation represents a critical target for quality breeding [1,2,3]. These compounds impart the characteristic orange-red coloration to wolfberry fruits and function as potent antioxidants and vitamin A precursors [4]. Carotenoids serve as the primary pigments responsible for the vibrant orange-red coloration of wolfberry and also function as potent antioxidants (zeaxanthin and its dipalmitate ester) and vitamin A precursors (β-carotene) [5,6]. Dried wolfberry exhibited high total carotenoid content, ranking among the highest observed in common fruits and vegetables, with red wolfberry averaging 233.04 μg·g−1 [7]. Elucidating the functional genes involved in carotenoid biosynthesis, their expression patterns, and how environmental stresses like salinity modulate this pathway has become a central strategy for developing novel wolfberry varieties with enhanced carotenoid content and improved salt tolerance.
Carotenoid biosynthesis occurs through a series of enzymatic reactions in plastids and chloroplasts, mediated by multiple key genes including PSY (phytoene synthase), PDS (phytoene desaturase), ZDS (ζ-carotene desaturase), LCYB (lycopene β-cyclase), LCYE (lycopene ε-cyclase), BCH (β-carotene hydroxylase), and ZEP (zeaxanthin epoxidase), which function through coordinated expression and precise regulation [8,9]. These genes encode rate-limiting enzymes and branch-point enzymes that play pivotal roles in the carotenoid biosynthetic pathway. PSY, as the rate-limiting enzyme catalyzing the first reaction, controls the initiation rate of the entire pathway. LCYB and LCYE competitively regulate the direction of cyclization reactions at branch points. Through dynamic changes in expression levels, fine-tuned enzymatic activities, and intricate interactions, these enzymes collectively determine carbon flux directionality and ultimately influence the composition and proportions of final products [10,11]. Till now, some key carotenoid genes in L. barbarum have been preliminarily identified [12,13]. Comprehensive characterization of the entire gene family and investigations into their dynamic regulatory networks across developmental stages or under various cultivars/environmental conditions remain limited.
In higher plants, the biosynthesis process of carotenoids is controlled by a complex multi-level regulatory network, which consists of three interrelated levels, including environmental factors (light intensity, temperature fluctuations, abiotic stresses), endogenous signals (abscisic acid (ABA) and sugar signaling), and transcriptional regulation of biosynthetic genes [14,15,16,17]. For L. barbarum, when attempting to achieve important medicinal and edible crop-cultivation, adverse conditions such as secondary salinization or saline-alkali soils are frequently encountered. Salt stress hinders normal growth and development and also significantly alters the biosynthesis and accumulation process of carotenoids by interfering with these regulatory networks, ultimately affecting the quality and yield of the fruits of L. barbarum [18]. Treatment with 300 mM NaCl resulted in the identification of 1396 differentially expressed genes (DEGs) and 71 differentially accumulated metabolites (DAMs) in mature wolfberry fruits. Integrated metabolomic and transcriptomic analyses further revealed that salt stress markedly promotes carotenoid esterification (zeaxanthin dipalmitate formation) and flavonoid glycosylation, primarily through the significant upregulation of oxidoreductases, hydrolases, and modification enzymes such as acyltransferases and glycosyltransferases [19]. A central mediator in this process is reactive oxygen species (ROS), which rapidly accumulate under salt stress. Beyond their well-known cytotoxic effects, ROS function as essential secondary messengers that activate downstream defense pathways, including mitogen-activated protein kinase (MAPK) cascades and transcriptional networks regulating antioxidant metabolism [20,21]. Despite preliminary identification of several carotenoid biosynthetic genes in L. barbarum, comprehensive insights into their genomic organization, regulatory mechanisms, and stress-responsive expression patterns remain limited. In particular, how these genes are dynamically coordinated under salt stress to balance precursor supply, pigment biosynthesis, and photoprotective metabolite accumulation is still poorly understood.
In this study, we systematically characterized the structural genes involved in carotenoid biosynthesis in L. barbarum through an integrated transcriptomic and bioinformatic approach. Comprehensive analyses were performed to elucidate gene structures, conserved motifs, chromosomal distribution patterns, and cis-acting regulatory elements. Then, tissue-specific expression profiles across different developmental stages were delineated. The investigation primarily focused on examining the expression dynamics of carotenoid biosynthetic genes under salt stress conditions. These findings provide potential support for molecular breeding strategies aimed at enhancing both fruit quality and stress tolerance in wolfberry.

2. Materials and Methods

2.1. Plant Materials and Treatment Methods

The study utilized Lycium barbarum var. auranticarpum ‘Ningqi No. 1’ plants cultivated at the National Wolfberry Engineering Technology Research Center in Yinchuan, Ningxia (37°53′ N, 105°72′ E). Uniformly grown, disease-free plants were acclimated for 7 days in growth chambers under controlled conditions: temperature 25 ± 2 °C, photoperiod 16 h light/8 h dark, light intensity 300 μmol·m−2·s−1, and relative humidity 60%. Following acclimation, plants were subjected to salt stress by root immersion in nutrient solution containing 300 mM NaCl [19,22]. Leaf samples were collected at seven time points (0, 1, 3, 6, 9, 12, and 24 h), with three biological replicates per time point. Samples were immediately frozen in liquid nitrogen and stored at −80 °C for subsequent analysis.

2.2. Data Sources and Candidate Gene Screening

The L. barbarum genome data were obtained from the Wolfberry Genome Database (WGBD, version 2024; https://cosbi7.ee.ncku.edu.tw/Wolfberry/) [23]. Candidate carotenoid biosynthetic genes were first identified by BLASTP searches (v2.12.0, NCBI, default parameters) against the L. barbarum protein database, using well-characterized carotenoid-related genes from Arabidopsis thaliana and Solanum Lycopersicon (PSY, PDS, ZDS, LCYB, LCYE, BCH, ZEP) as queries (Table S1). BLASTP was selected rather than iterative tools such as PSI-BLAST or DELTA-BLAST, as the aim was to identify reliable close homologs within the Solanaceae, rather than more distant evolutionary relationships. Only protein-coding sequences (CDS-derived protein sequences) were employed for the searches to ensure functional homology at the amino acid level. To confirm the identity of candidate sequences, conserved domain verification was performed using HMMER (v3.3.2) with hidden Markov model (HMM) profiles from the Pfam database (v35.0) [24,25].

2.3. Gene Structure and Chromosomal Localization Analysis

Gene structure information was obtained from the L. barbarum genome annotation file (GFF3) downloaded from the Wolfberry Genome Database. Exon–intron organizations were visualized using GSDS 2.0 (http://gsds.gao-lab.org/) with default parameters. Chromosomal locations of the identified genes were determined using the GFF3 coordinates and visualized in TBtools (v1.120) with the “Gene Location Visualize” function. For TBtools analysis, default visualization settings were applied, and no additional adjustments were made to scaling or chromosome size parameters [26].

2.4. Protein Domain Prediction and Functional Annotation

Conserved domain prediction was performed using the NCBI Conserved Domain Database (CDD, accessed June 2024) and the Pfam database (v35.0). Domain hits were retained only if they met the following criteria: e-value ≤ 1 × 10−5, minimum domain coverage ≥ 50% of the query sequence length, and identity ≥ 30%. Domain architectures were then illustrated using the “Simple Protein Motif View” function in TBtools (v1.120).

2.5. Synteny Analysis

Genome assemblies and annotation files (GFF3) of L. barbarum, A. thaliana, and S. lycopersicon were retrieved from publicly available databases: L. barbarum from the Wolfberry Genome Database, A. thaliana from TAIR10 release (https://www.arabidopsis.org), and S. lycopersicon from the Sol Genomics Network (SGN, ITAG4.1 release, https://solgenomics.net/). For synteny analysis, protein sequences were first compared using BLASTP (v2.12.0, NCBI) with an e-value cutoff of 1 × 10−5, maximum target sequences = 5, and default scoring parameters. The resulting BLASTP alignment files and corresponding GFF3 annotations were formatted as input for MCScanX. Collinear blocks were defined using MCScanX (as implemented in TBtools v1.120), applying a minimum of 5 homologous gene pairs per block as the threshold for synteny. Results were visualized with the Advanced Circos and Multiple Synteny Plot functions in TBtools [27].

2.6. Promoter cis-Element Prediction

For promoter analysis, the 2000 bp upstream sequences of each structural gene were extracted from the genome assembly using GFF3 annotations. To avoid erroneous annotation, promoter regions were cross-checked to ensure they did not overlap with coding sequences of neighboring genes. cis-acting elements were identified using the Plant CARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Identified motifs were grouped into functional categories (light-responsive, stress-responsive, hormone-responsive, and circadian-related elements) following the PlantCARE classification system. Visualization of the distribution and frequency of cis-elements was performed with TBtools (v1.120) [28]. PlantCARE was selected because of its broad use in plant genomic studies and its clear functional categorization of regulatory elements, which met the needs of this study.

2.7. Expression Profile Analysis and Dynamic Analysis of Stress Response Expression

To systematically characterize the expression profiles of carotenoid structural genes, we conducted integrated analyses using both tissue-specific expression data and time-course transcriptomes under salt stress conditions. For both the tissue-specific and developmental datasets, three independent biological replicates were analyzed per tissue or stage. Replicates were obtained from different individual plants of the same cultivar grown under identical environmental conditions. For RNA-seq quality control, raw reads were first assessed using FastQC (v0.11.9), and adapter sequences and low-quality bases (Q < 20, read length < 50 bp) were trimmed with Trimmomatic (v0.39). Cleaned reads were then used for downstream alignment. For tissue-specific expression analysis, we retrieved the raw sequencing reads (FASTQ files) from the public dataset PRJNA845109. All samples were processed through the same analysis pipeline (FastQC → Trimmomatic → Hisat2 → String Tie) to ensure comparability [29]. For the salt stress experiment, clean reads from leaf samples at seven time points (0, 1, 3, 6, 9, 12, and 24 h after 300 mM NaCl treatment) were aligned to the L. barbarum reference genome using Hisat2 (v2.2.1) with default parameters, except that the maximum mismatch allowance was set to two per read and only uniquely mapped reads were retained. Transcript assembly and quantification were then performed using String Tie (v2.1.4) with default settings. Expression levels of carotenoid structural genes were normalized as FPKM values. To explore expression dynamics, normalized FPKM values were standardized and subjected to K-means clustering implemented in R (v3.2.2). The optimal number of clusters (K) was determined by testing a range of values (K = 2–10) and evaluating both the within-cluster sum of squares (SSE, elbow method) and the silhouette index. The K value that minimized SSE while maximizing the silhouette index was selected for downstream analysis. Final clustering and visualization of expression patterns were carried out using TBtools (v1.120) [30]. Differential expression analysis was performed using DESeq2 (v1.36.0). Genes were considered differentially expressed if they met both criteria: a false discovery rate (p-value < 0.05) and an absolute fold-change ≥ 2 (|log2FC| ≥ 1).

3. Results

3.1. Analysis of Physicochemical Properties of Carotenoid Biosynthetic Proteins in Wolfberry

Through systematic screening, we identified and analyzed the molecular characteristics of 17 structural genes involved in carotenoid biosynthesis in L. barbarum (Table 1). The results demonstrated substantial variation in the encoded proteins, with amino acid lengths ranging from 292 to 1099 residues and molecular weights spanning 32,712.94 to 122,847.17 Da. Bioinformatic analysis revealed that most genes encoded proteins with relatively high theoretical isoelectric points (pI), suggesting predominantly alkaline properties. Stability assessment indicated that LbaCYP97C11 exhibited the lowest instability index (35.27), implying superior structural stability. Proteins encoded by the genes with elevated aliphatic indices, particularly LbaDXS2 (92.98) and LbaDXS1 (95.71), were predicted to demonstrate enhanced stability in membrane or membrane-associated structures. Subcellular localization predictions indicated predominant targeting to chloroplasts, cytoplasm, and plasma membranes, consistent with potential involvement in compartmentalized metabolic processes and functional activities across different cellular domains.

3.2. Structural and Functional Analysis of Carotenoid Biosynthetic Genes

Figure 1 illustrated the exon–intron organization of the 17 candidate genes. The analysis revealed considerable variation in exon numbers, ranging from a minimum of 5 exons in genes such as LbaBCH2 and LbaZISO to a maximum of 18 exons in the most structurally complex gene, LbaPDS. Several genes including LbaLCYE, LbaCRTISO, and LbaCYP97A29 contained more than 10 exons, suggesting potentially sophisticated splicing regulation and functional complexity. Furthermore, substantial differences in gene length were observed, with LbaPDS exhibiting an exceptionally long transcriptional unit (>45 kb), in contrast to the compact structures of genes such as LbaBCH1 and LbaZEP. Conserved domain analysis of the encoded proteins (Figure 2) identified at least one characteristic functional domain in each protein, with a total of 18 distinct domain types detected. Frequently observed domains included PLN02463, PLN02582 superfamily, and PLN02612 superfamily, which represent conserved modules associated with carotenoid biosynthetic enzymes. The LbaDXS1 and LbaDXS2 contained the PLN02582 superfamily domain, consistent with their established roles in chlorophyll and carotenoid precursor biosynthesis. LbaPDS and LbaZDS shared the PLN02487 domain, indicative of their involvement in dehydrogenation reactions. LbaZEP uniquely possessed the PLN02927 superfamily domain, characteristic of cyclization and hydroxylation activities. Notably, some genes encoded proteins with multiple domains (two distinct domains in LbaPDS), suggesting potential multifunctional roles or participation in multiple reaction steps. These domain architecture differences provide valuable insights for subsequent functional characterization studies.

3.3. Genomic Distribution and Evolutionary Analysis of Carotenoid Biosynthetic Genes

Based on genome annotation and mapping, we identified 17 carotenoid-related structural genes distributed across 10 chromosomes (Chr01-Chr12, except Chr05 and Chr09) in L. barbarum (Figure 3). These genes showed distinct chromosomal distribution patterns, with Chr08 and Chr10 containing the most genes (3 each, accounting for 18% of the total). Chr01, Chr03 and Chr12 each contained 2 genes, while the remaining chromosomes had 1 gene each. To examine the evolutionary conservation of these genes, we constructed synteny maps between L. barbarum and two reference species: A. thaliana and S. lycopersicum (Figure 4). The analysis revealed 8 syntenic blocks between L. barbarum and A. thaliana, with high conservation between A. thaliana Chr05 and multiple L. barbarum chromosomal regions (Chr03, Chr04, Chr12). Total of 18 syntenic blocks between L. barbarum and S. lycopersicum, showing closer evolutionary relationships, particularly on Chr01, Chr03 and Chr08. Half of the carotenoid structural genes (9/17) were located in these syntenic regions, indicating strong conservation within Solanaceae. Several genes formed clusters on chromosomes (Lba08g01705, Lba08g01811 and Lba08g02019 on Chr08; Lba10g01149 and Lba10g01158 on Chr10), suggesting their potential co-regulation in carotenoid biosynthesis.

3.4. Analysis of cis-Regulatory Elements of Carotenoid Biosynthetic Genes in L. barbarum

To investigate the transcriptional regulation of carotenoid biosynthesis-related genes, we systematically predicted and analyzed the cis-acting elements in their promoter regions (2000 bp upstream sequences), with results shown in Figure 5. The figure displays various types of cis-acting elements and their distribution in the promoter regions of 17 carotenoid biosynthesis-related genes. These elements mainly include regulatory elements related to hormone response, stress response, light response, and circadian rhythm. Light-responsive elements were widely detected in all analyzed promoters, indicating that light signals may play a dominant role in regulating carotenoid biosynthesis genes. The genes (LbaPDS, LbaLCYB, and LbaCRTISO) also contain elements related to low-temperature response, jasmonic acid response, and abscisic acid response, suggesting these genes may play important roles under stress conditions. Additionally, elements related to auxin, gibberellin, and salicylic acid were detected in the genes (LbaPSY1, LbaPSY2, and LbaZDS), indicating that hormone signals may also be involved in regulating the expression of these genes. Elements related to circadian rhythm regulation were only found in the two genes (LbaZEP and LbaLCYE), suggesting that circadian regulation of carotenoid biosynthesis is relatively limited or specific.

3.5. Tissue-Specific and Developmental Expression Patterns of Carotenoid Biosynthetic Genes

The heatmap analysis revealed distinct expression profiles of the 17 carotenoid biosynthetic genes across various tissues and developmental stages in L. barbarum (Figure 6). During fruit maturation (S1–S5), a progressive upregulation of the genes was observed, LbaPSY1, LbaPDS, LbaZDS, and LbaBCH2, exhibiting peak expression at late developmental stages (S4–S5). In contrast, floral tissues (S1–S3) generally displayed lower expression levels, except for LbaPSY2 and LbaZEP, which showed significantly elevated expression during the S3 floral stage. Leaf tissues demonstrated particularly high expression of photoprotective genes (LbaZEP, LbaVDE, and LbaCYP97C11), consistent with their putative role in photosystem stabilization, whereas stem tissues maintained consistently low expression across all genes, suggesting minimal involvement of the carotenoid biosynthetic pathway in this tissue type. These tissue-specific and developmentally regulated expression patterns provide important insights into the spatial and temporal regulation of carotenoid biosynthesis in wolfberry berries.

3.6. Expression Dynamics of Carotenoid Synthesis Pathway Genes Under 300 mM NaCl Treatment

Transcriptional profiling of carotenoid biosynthetic genes under 300 mM NaCl treatment revealed a phased response pattern in L. barbarum (Figure 7). The metabolic pathway demonstrated sequential activation of distinct gene clusters corresponding to three temporal phases. Within 1–3 h post-treatment, immediate upregulation of upstream MEP pathway genes (DXS and GGPS) facilitated rapid precursor synthesis. During 6–12 h, coordinated induction of core biosynthetic genes (PSY, PDS, ZISO, ZDS, and CRTISO) promoted lycopene accumulation, with preferential activation of the β-branch through LYCB upregulation while LYCE expression remained stable. The late phase (9–24 h) featured sustained induction of xanthophyll cycle components (BCH1/2, ZEP, VDE, and NXS), enhancing production of photoprotective carotenoids (violaxanthin and neoxanthin), accompanied by moderate upregulation of α-carotene branch genes (CYP97A29 and CYP97A11) contributing to lutein biosynthesis. This temporally coordinated expression cascade illustrated a sophisticated adaptive strategy wherein sequential pathway activation ensures both precursor availability and oxidative stress mitigation under saline conditions.

4. Discussion

Carotenoid biosynthesis was regulated by multiple genes, with key enzymes, such as PSY and PDS, playing pivotal roles throughout the metabolic pathway [31,32]. Through comprehensive genomic analysis, we identified 17 genes associated with carotenoid biosynthesis in L. barbarum. These genes are distributed across 10 chromosomes, with two distinct gene clusters observed on Chr08 (LbaCYP97C11-LbaCRTISO-LbaLCYB) and Chr10 (LbaCYP97A29-LbaVDE), potentially resulting from localized gene duplication events [33]. The strong syntenic relationship with S. lycopersicum (18 syntenic blocks), significantly higher than that with A. thaliana (8 blocks), confirms the evolutionary conservation among Solanaceae species [33,34]. This conservation reflects the close phylogenetic relationship within Solanaceae and suggests functional implications. Gene clusters like LbaCYP97C11–LbaCRTISO–LbaLCYB on Chr08 and LbaCYP97A29–LbaVDE on Chr10 may have arisen from recent duplication events, providing opportunities for sub functionalization or even neofunctionalization [35]. Promoter analysis revealed a significant enrichment of light-responsive elements (G-box) across all the 17 genes, consistent with the well-established model of light-mediated regulation of carotenoid synthesis [36]. Promoter cis-element analysis revealed enrichment of light-responsive motifs (G-box), consistent with experimental studies showing that G-box–binding transcription factors directly regulate PSY expression in Arabidopsis and tomato [37]. Stress-related motifs, including ABRE and DRE, were also identified, in line with reports that ABA- and drought-responsive cis-elements modulate carotenoid pathway genes in carrot and other crops [9,16]. Furthermore, circadian-associated elements such as the CCA1-box have been linked to diurnal regulation of carotenoid metabolism in algae and higher plants [38]. The continuous advancement of transcriptome sequencing technologies has established this approach as an indispensable tool for plant genomic research and crop genetic improvement [39,40,41]. In the present study, we employed transcriptome sequencing to systematically analyze the expression patterns of the 17 carotenoid structural genes in L. barbarum. The biosynthetic pathway of carotenoids showed responsiveness to the environment and also exhibits significant tissue specificity [31]. Our organ-specific and developmental expression profiling revealed that the genes encoding biosynthetic enzymes (PSY1, PDS, and ZDS) exhibited significantly enhanced expression during mid-to-late fruit developmental stages (S3–S5). This expression pattern correlated temporally with fruit color intensification [12]. Comparative analysis suggested that the elevated expression of these genes directly drives the accumulation of nutritionally important carotenoids (β-carotene, lutein, and zeaxanthin), thereby influencing both nutritional quality and sensory characteristics of the fruits [42]. This regulatory mechanism contrasts with that observed in tomato fruit ripening, where sustained upregulation of PSY, PDS, ZDS, and CRTISO coupled with minimal expression of LCY and B-LCY genes leads to predominant lycopene accumulation [43,44]. Floral tissues generally maintained lower expression levels of carotenoid biosynthetic genes, with the exception of PSY and ZEP which showed peak expression during stage III of flower development, consistent with previous reports [45]. In leaf tissues, genes of the xanthophyll cycle (ZEP, VDE, and CYP97C11) were predominantly expressed, consistent with their role in photoprotection by maintaining photosystem stability under high light conditions [46]. This tissue-specific enrichment is particularly relevant to our findings under salt stress, where the late-phase induction of the same genes facilitated the accumulation of photoprotective carotenoids (violaxanthin and neoxanthin). Together, these results indicate that the xanthophyll cycle genes safeguard photosynthetic tissues under light stress and act as critical components of the adaptive antioxidant response when plants encounter salinity.
Carotenoids function as accessory pigments in photosynthesis, capturing light energy and transferring it to chlorophylls under normal conditions. Under abiotic stress, their role in photoprotection becomes particularly critical, as specific carotenoids such as zeaxanthin, lutein, and β-carotene dissipate excess energy, quench reactive oxygen species, and stabilize photosynthetic complexes [47,48,49]. Wolfberry is considered a potential pioneer species for saline–alkali land improvement due to its inherent salt tolerance. Nevertheless, high salinity strongly inhibits its growth and photosynthesis. Here, a 300 mM NaCl treatment was applied to simulate the severe salt stress typical of saline–alkali soils in Ningxia [18]. Our findings demonstrated that 300 mM NaCl treatment elicited a temporally coordinated and modular transcriptional response in the carotenoid biosynthetic pathway of L. barbarum. During the early stress phase (1–3 h), rapid upregulation of the upstream MEP pathway genes (DXS and GGPS), encoding the rate-limiting enzymes, ensured sufficient precursor supply for downstream metabolism. The intermediate phase (6–12 h) was characterized by sustained induction of core biosynthetic enzymes (PSY, PDS, ZISO, ZDS, and CRTISO), which collectively enhanced carotenoid production and specifically promoted lycopene accumulation, with preferential activation of the β-branch through LCYB upregulation while LCYE expression remained stable. This β-branch preference has adaptive significance, as β-carotene acts as a precursor for photoprotective xanthophylls (zeaxanthin, violaxanthin) and for the phytohormone abscisic acid (ABA). This mid-phase activation in L. barbarum under 300 mM NaCl closely parallels findings in Daucus carota, where 300 mM NaCl treatment significantly elevated the expression of DcPSY1, DcPSY2, DcZDS1, DcCRT1, DcCRT2, DcLCYB, and DcLCYE, along with increased carotenoid accumulation [50]. These results highlighted the central role of this pathway in redirecting carbon flux under salt stress, consistent with previous findings in tomato [51]. In the late stress phase (12–24 h), pronounced upregulation of downstream photoprotective and antioxidant-related genes (ZEP, VDE, and NXS) facilitated the biosynthesis of highly active antioxidant metabolites, including zeaxanthin, violaxanthin, and neoxanthin, thereby improving reactive oxygen species (ROS) scavenging capacity. This “precursor-prioritized to product-accumulation” phased expression strategy suggested the molecular mechanisms by which L. barbarum dynamically maintains carotenoid metabolic homeostasis under salt stress [20]. The time-series design in this study was suitable for analyzing the early transcriptional responses under salt stress, but it did not include a post-stress recovery phase. Consequently, our understanding of transcriptional resilience and metabolic reprogramming remains limited. Future studies incorporating recovery sampling after salt treatment will provide a more comprehensive view of the adaptive and restorative mechanisms of wolfberry under saline conditions.
Unlike previous studies that mainly identified carotenoid genes or described static expression patterns, this work provides new insights. We performed a more detailed promoter analysis, revealing enrichment of light- and stress-responsive elements that indicate multi-layered regulation. We also demonstrated clear phased expression dynamics under salt stress, showing a cascade from precursor supply to antioxidant product accumulation. Our findings advanced the understanding of this regulatory network and also provided a potential foundation for improving stress tolerance and nutritional quality through gene editing or molecular breeding. The upregulated genes identified in this study (PSY, ZDS, and BCH1) may serve as potential targets for future genetic improvement of L. barbarum aimed at enhancing stress resistance and carotenoid accumulation. In addition, CRISPR/Cas-based genome editing offers the possibility of fine-tuning key structural genes or promoter motifs to optimize flux through the carotenoid pathway. Such approaches would accelerate the development of L. barbarum cultivars with improved nutritional quality and tolerance to saline–alkali conditions. It should be noted that the 300 mM NaCl treatment represents an extreme salinity condition. Similar concentrations have been applied in wolfberry germplasm studies to evaluate salt tolerance diversity [52]. This level likely exceeds typical field conditions in Ningxia saline–alkali soils. Our results should be interpreted as an extreme stress model, and future work with soil ion data and moderate NaCl gradients will better reflect ecological relevance.

5. Conclusions

This study systematically analyzed the expression regulatory characteristics of the structural genes in the carotenoid synthesis pathway of wolfberry under salt stress and constructed a dynamic regulatory network with temporal response and modular distribution. We identified 17 core structural genes, distributed in clusters on chromosomes Chr08 (LbaCYP97C11, LbaCRTISO, LbaLCYB) and Chr10 (LbaCYP97A29, LbaVDE), exhibiting pronounced synteny with tomato (18 collinear pairs), which reflects evolutionary conservation within Solanaceae. Promoter cis-element analysis revealed pervasive enrichment of light-responsive (G-box) and stress-responsive motifs (ABRE/DRE), providing a molecular basis for environmental signal integration. 300 mM NaCl elicits a temporally phased transcriptional cascade: During the early stress phase (1–3 h), genes encoding MEP pathway enzymes (DXS, GGPS) are rapidly induced to augment precursor supply. In the mid-phase (6–12 h), core synthases (PSY, PDS, ZDS) are upregulated, channeling metabolic flux toward lycopene and preferentially activating the β-branch via LYCB. The late phase (12–24 h) features enhanced expression of xanthophyll cycle genes (BCH, ZEP, VDE), facilitating violaxanthin and neoxanthin accumulation to mitigate oxidative damage. This coordinated “precursor-to-protectant” strategy demonstrates metabolic adaptation to salinity.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11101149/s1, Table S1: Reference carotenoid biosynthetic genes used as queries for BLASTP searches.

Author Contributions

Conceptualization, A.-S.X., Y.Y., Z.-H.H. and J.Z.; methodology, Y.Y. and Z.-H.H.; investigation, Z.-H.H. and Y.Y.; data curation, Z.-H.H., L.-X.W., N.Z. and C.C.; project administration, A.-S.X., Y.Y. and J.Z.; writing—original draft preparation, Z.-H.H.; writing—review and editing, Z.-H.H., A.-S.X. and J.Z. All authors have read and agreed to the published version of the manuscript.

Funding

The research was supported by the Key Research and Development projects of Ningxia Hui Autonomous Region (No. 2022BBF02008), the Key Research and Development Program of Suqian (No. L202403), the Priority Academic Program Development of Jiangsu Higher Education Institutions Project (No. PAPD), and the Bioinformatics Center of Nanjing Agricultural University.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Figure 1. Exon–intron structure analysis of the carotenoid structural genes in wolfberry. The green squares represented exons, and the black lines represented introns. The gene names were listed on the left side.
Figure 1. Exon–intron structure analysis of the carotenoid structural genes in wolfberry. The green squares represented exons, and the black lines represented introns. The gene names were listed on the left side.
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Figure 2. Analysis of conserved domains of the protein encoded by the structural gene of carotenoids in wolfberry. Each color represented a different conservative domain, and the right legend marked the domain names of each structure.
Figure 2. Analysis of conserved domains of the protein encoded by the structural gene of carotenoids in wolfberry. Each color represented a different conservative domain, and the right legend marked the domain names of each structure.
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Figure 3. The localization of the structural genes of carotenoids biosynthesis on chromosomes in wolfberry. The color bars represented the heat maps of each chromosome, with colors ranging from blue to red indicating different gene densities.
Figure 3. The localization of the structural genes of carotenoids biosynthesis on chromosomes in wolfberry. The color bars represented the heat maps of each chromosome, with colors ranging from blue to red indicating different gene densities.
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Figure 4. Co-linearity analysis of carotenoid structural genes in L. barbarum, A. thaliana and S. lycopersicum. The red lines indicated the homologous collinear fragments corresponding to the structural genes of carotenoids, and the background showed the collinear relationship of the entire genome.
Figure 4. Co-linearity analysis of carotenoid structural genes in L. barbarum, A. thaliana and S. lycopersicum. The red lines indicated the homologous collinear fragments corresponding to the structural genes of carotenoids, and the background showed the collinear relationship of the entire genome.
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Figure 5. Predictive analysis of cis-acting elements of gene promoters related to carotenoid synthesis in L. barbarum.
Figure 5. Predictive analysis of cis-acting elements of gene promoters related to carotenoid synthesis in L. barbarum.
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Figure 6. Heatmap of the expression of genes related to carotenoid synthesis in wolfberry at different tissue and fruit development stages.
Figure 6. Heatmap of the expression of genes related to carotenoid synthesis in wolfberry at different tissue and fruit development stages.
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Figure 7. Heatmap of the expression profiles of carotenoid biosynthesis pathways and the genes under 300 mM NaCl treatment. Each color block represented a time point (0 h, 1 h, 3 h, 6 h, 9 h, 12 h, 24 h), and the color changed from blue (down-regulation) to red (up-regulation) reflect the trend of expression levels.
Figure 7. Heatmap of the expression profiles of carotenoid biosynthesis pathways and the genes under 300 mM NaCl treatment. Each color block represented a time point (0 h, 1 h, 3 h, 6 h, 9 h, 12 h, 24 h), and the color changed from blue (down-regulation) to red (up-regulation) reflect the trend of expression levels.
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Table 1. Sequence information of Carotenoid structural genes in L. barbarum..
Table 1. Sequence information of Carotenoid structural genes in L. barbarum..
Gene NameGene NumberNumber of Amino AcidMolecular WeightTheoretical pIInstability IndexAliphatic IndexGrand Average of HydropathicitySubcellular Location
LbaBCH1Lba01g0131930433,996.519.2246.6982.170.001Plasma membrane
LbaNXSLba01g0262270680,304.389.2846.6688.5−0.196Chloroplast
LbaDXS1Lba02g0142642946,494.118.2545.1995.71−0.092Cytosol
LbaBCH2Lba03g0150529232,712.948.8948.8988.9−0.03Chloroplast
LbaPDSLba03g031271099122,847.178.1547.7992.98−0.153Vacuolar membrane
LbaLCYELba04g0285552358,534.687.0344.3190.21−0.043Plasma membrane
LbaZDSLba06g0169554359,423.047.5239.3387.29−0.14Chloroplast
LbaZISOLba07g0202137342,133.819.130.7100.640.157Plasma membrane
LbaCYP97C11Lba08g0170554660,951.736.1935.2793.75−0.169Chloroplast
LbaCRTISOLba08g0181160466,427.47.8633.5391.99−0.095Cytosol
LbaLCYBLba08g0201950156,609.627.9835.989.46−0.15Chloroplast
LbaDXS2Lba10g0025457662,241.748.3942.9599.91−0.034Chloroplast
LbaCYP97A29Lba10g0114963971,390.935.6146.1389.11−0.184Cytosol
LbaVDELba10g0115847053,558.046.0243.3582.34−0.454Chloroplast
LbaPSY1Lba11g0232441246,499.078.0945.7184.51−0.312Chloroplast
LbaPSY2Lba12g0087444950,938.219.255.2881.92−0.379Chloroplast
LbaZEPLba12g0241266372,198.587.1431.5288.97−0.156Chloroplast
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Hu, Z.-H.; Wang, L.-X.; Zhang, N.; Chen, C.; Zhuang, J.; Yin, Y.; Xiong, A.-S. Expression Regulatory Mechanisms of the Key Structural Genes in the Carotenoid Biosynthesis Pathway Under Salt Stress of Lycium barbarum. Horticulturae 2025, 11, 1149. https://doi.org/10.3390/horticulturae11101149

AMA Style

Hu Z-H, Wang L-X, Zhang N, Chen C, Zhuang J, Yin Y, Xiong A-S. Expression Regulatory Mechanisms of the Key Structural Genes in the Carotenoid Biosynthesis Pathway Under Salt Stress of Lycium barbarum. Horticulturae. 2025; 11(10):1149. https://doi.org/10.3390/horticulturae11101149

Chicago/Turabian Style

Hu, Zhi-Hang, Li-Xiang Wang, Nan Zhang, Chen Chen, Jing Zhuang, Yue Yin, and Ai-Sheng Xiong. 2025. "Expression Regulatory Mechanisms of the Key Structural Genes in the Carotenoid Biosynthesis Pathway Under Salt Stress of Lycium barbarum" Horticulturae 11, no. 10: 1149. https://doi.org/10.3390/horticulturae11101149

APA Style

Hu, Z.-H., Wang, L.-X., Zhang, N., Chen, C., Zhuang, J., Yin, Y., & Xiong, A.-S. (2025). Expression Regulatory Mechanisms of the Key Structural Genes in the Carotenoid Biosynthesis Pathway Under Salt Stress of Lycium barbarum. Horticulturae, 11(10), 1149. https://doi.org/10.3390/horticulturae11101149

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