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Article

Genome-Wide Identification of Cucumber Lhc Genes’ Family and Their Expression Analysis

by
Yongmei Miao
1,* and
Kaijing Zhang
2
1
College of Biomedicine and Health, Anhui Science and Technology University, Chuzhou 233100, China
2
College of Agriculture, Anhui Science and Technology University, Chuzhou 233100, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(7), 736; https://doi.org/10.3390/horticulturae11070736
Submission received: 11 February 2025 / Revised: 15 June 2025 / Accepted: 18 June 2025 / Published: 25 June 2025
(This article belongs to the Special Issue The Role of Plant Growth Regulators in Horticulture)
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Abstract

Light-harvesting chlorophyll a/b-binding (Lhc) proteins are integral membrane proteins that bind to pigment molecules, playing a critical role in photosynthetic processes, including light energy harvesting and transfer. To investigate the role of the Lhc gene family in cucumber (Cucumis sativus L), genome-wide identification of CsLhc gene family members and analysis of their regulatory networks were carried out using bioinformation and molecular biology research methods at Anhui Science and Technology University. The results indicated that the Lhc family consisted of 21 members, being categorized into five subfamilies: Lhca, Lhcb, CP24, CP26, and CP29. The gene structure and motifs within each subfamily are generally conserved. CsLhcs are distributed on seven chromosomes, including one pair of tandem duplicates and two pairs of segmental duplicates. Six CsLhcs exhibit eight linear relationships with seven AtLhcs, and one CsLhc shows a syntenic relationship with one OsLhc. Analysis of the cis-acting elements in CsLhc promoters revealed their potential involvement in stress responses. Transcriptome data indicated that CsLhcs are minimally expressed in male flowers and roots, but highly expressed in other organs. Analysis of stress response processes revealed that all Lhc genes participate in at least one stress response. Five Lhc genes were confirmed to appear to have expression change using qPCR analysis under high temperature and salt stress. Particularly, under downy mildew, root-knot nematode stresses, and blight stress, up-regulated Lhc genes were the most abundant ones, indicating that the Lhc family acts as a significant role in the growth and development of cucumber. These results provide valuable insights for further understanding the characteristics of the CsLhc family and analyzing the function of the Lhc family in cucumber resistance to biotic/abiotic stresses and in molecular breeding.

1. Introduction

Chlorophyll in green plants participates in photosynthesis by capturing light energy and transferring it into chemical energy, which is essential for cellular metabolic processes [1]. Light-harvesting complex (LHC) superfamily proteins can bind to chlorophyll and carotenoid to form pigment–protein complexes, which act as antenna proteins and fulfill crucial roles in photosynthesis, photoprotection of PSII micro-tissue, and the attenuation of oxidative stress processes [2,3,4,5,6]. The LHC superfamily possessing plant-specificity comprises four families: Lhc, Lil, PsbS, and FCII [7].
Light-harvesting chlorophyll a/b-binding (Lhcs) are cyst-like membrane proteins, which are the most abundant in nature [8]. The Lhc family primarily consists of two evolutionary groups, Lhca and Lhcb, which are respectively associated with photosystem I (PSI) and photosystem II (PSII) [9]. The interplay between PSI and PSII is a key in efficient photosynthesis: Water is decomposed by PSII to release high-energy electrons and oxygen, and PSI receives the high-energy electrons generated by PSII to NADP+ to generate NADPH for organic matter production [10]. It has also been proven that the Lhc family is involved in the regulation and distribution of energy between PSI and PSII, repair of light-induced damage in the photosynthetic system, response to many stresses, and nitrogen utilization [11,12,13]. The Lhc family has been analyzed in several important plants, including rice [14], Arabidopsis [14], cassava [15], Gossypium hirsutum [9], castor bean [16], kiwifruit [17], apple [3], and other species. In-depth research on the Lhc family has shown that it can respond to a variety of stresses. For example, in A. thaliana, AtLhcb members regulate ABA (abscisic acid)-induced stomatal movement [18], seed germination and growth, as well as plant adaptation to environmental changes [19,20]. Additionally, AtLhcb1 and AtLhcb2 exhibit distinct but complementary functions in the phosphorylation-driven state transition of light harvesting in A. thaliana [21]. In Oryza sativa, iron deficiency significantly reduces the expression of OsLhca1/2/3/4, leading to a substantial decrease in chlorophyll content and photosynthetic efficiency [22]. In Apium graveolens, the up-regulated expression of AgLhcb1 increased photosynthetic efficiency, suggesting its potential use as a marker for estimating photosynthetic rate [23]. The above results indicate that Lhc genes in plants play a significant role in coping with adversity of all kinds.
Cucumber is a globally consumed vegetable [24], and is popular for being low in calories, high in water, and rich in vitamins and minerals. The cucumber genome was sequenced as early as 2009 [25]. Utilizing high-quality genomic information, a large number of gene families have been identified in the cucumber genome, including WRKY [26], MADS-box [27], NBS [28], bZIP [29], and others. However, study on the identification and function of the Lhc family in cucumber has not yet been reported, limiting the utilization of their biological functions.
Therefore, in this study, cucumber genome bioinformatics were employed to identify the Lhc family and analyze the physical and chemical characteristics, chromosomal localization, conserved sequences, gene structure, phylogenetic relationships, promoter cis-acting elements, and upstream and downstream regulatory genes of its members. The tissue-specific expression of the CsLhc family and its expression patterns in stress response processes were also analyzed using cucumber transcriptome sequencing data, aiming to explore the effect of the Lhc family in response to biotic and abiotic stresses, and provide a foundation for future in-depth studies on Lhc function and for molecular breeding of stress resistance in cucumber.

2. Materials and Methods

2.1. Identification and Chromosomal Localization of Lhc Family in Cucumber

The HMM model file (PF00504) for the Lhc family was downloaded from the Pfam database (http://pfam.xfam.org/ (accessed on 14 October 2024)) [30], and the cucumber ChineseLong_V3 proteome sequence (http://cucurbitgenomics.org/ftp/genome/cucumber/Chinese_long/v3/ (accessed on 14 October 2024)) was downloaded from the Cucurbit Genome Database (http://www.icugi.org/ (accessed on 14 October 2024)). A local protein database was constructed, and potential Lhc IDs (E < 1 × 10−5) were screened from cucumber protein database using the hmmsearch program of the HMMER software package (version 3.0) [31]. The sequence information for candidate proteins was extracted using Perl scripts. The potential Lhc sequences were then verified for structural domains using the online tools Pfam and SMART (http://smart.embl.de/smart/batch.pl (accessed on 15 October 2024)) [32]. Sequences containing Lhc structural domains were selected to finalize Lhc family members in cucumber. Information on Arabidopsis Lhc family members was obtained based on a previous identification study [14] and downloaded from Arabidopsis genome website (https://www.Arabidopsis.org/ (accessed on 15 October 2024)). Physicochemical characteristics, including amino acid amount, molecular weight, isoelectric point, instability coefficient, fat coefficient, and hydrophilicity average were analyzed for cucumber Lhc family members using the ExPASy online tool (https://web.expasy.org/protparam/ (accessed on 15 October 2024)). Using Plant-mPLoc online tool (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/ (accessed on 16 October 2024)) [33], we predicted subcellular localization of cucumber Lhc. Lhc gene family was mapped across chromosomes using TBtools software (version 2.0) [34].

2.2. Characterization and Phylogenetic Analysis of Cucumber Lhc Family

Based on studies of Lhc families in Arabidopsis and rice [14], the proteins sequences of 21 Arabidopsis Lhcs and 15 rice Lhcs were downloaded. Using the MEME online software (version 5.5.8, http://meme-suite.org/ (accessed on 16 October 2024)), conserved motifs of Lhc proteins family in Arabidopsis, rice, and cucumber were analyzed, with the following parameters: number of matches is 10; optimal match length is 6–100 amino acids [35]. Phylogenetic trees of Lhc family in Arabidopsis, rice, and cucumber were constructed using MEGA 7 software with the neighbor-joining method. The following parameters were applied: Poisson model, pairwise deletion, and 1000 bootstrap replicates [36]. Phylogenetic trees were visualized and optimized using EvolView (https://www.evolgenius.info/evolview-v2/ (accessed on 16 October 2024)) [37]. Promoter elements of Lhc family members in cucumber were analyzed using PlantCare online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 17 October 2024)) [38].

2.3. Collinearity Analysis of Cucumber Lhc Family

To understand the interspecific collinearity of cucumber Lhc family with Arabidopsis and rice, the collinearity analysis was carried out using MCScanX software [39], and visualized using Circos software (version 0.69-9) [40].

2.4. Re-Analysis of Cucumber Transcriptome Sequencing Big Data Based on RNA-Seq

The cucumber transcriptome sequencing data, published in the SRA database (https://www.ncbi.nlm.nih.gov/sra (accessed on 18 October 2024)), were downloaded. The downloaded SRA data were converted to Fastq format using fasterq-dump (version 2.11.0). The quality of the Fastq data was assessed using FastQC software (version 0.12.1) [41]. Low-quality sequences and adapter regions in the Fastq data were removed using Trimmomatic software (version 0.39) [42], resulting in filtered clean data. The index of the cucumber ChineseLong_V3 genome was constructed using STAR software (version 2.7.10a). The filtered clean data were aligned to the cucumber ChineseLong_V3 genome, generating SAM files. The SAM files were then converted to sorted BAM files using SAM tools software (version 1.15) [43]. Gene expression data were estimated using StringTie software (version 2.2.1) [44]. Finally, differential gene expression was analyzed using DESeq software (version 2), based on the count data for every gene [45].

2.5. Expression Analysis of Lhc Family in Cucumber

The transcriptome sequencing project PRJNA80169 [46] of different cucumber tissues, high temperature (PRJNA634519) [47], low temperature PRJNA80169) [48], salt (PRJNA511946) [49], waterlogging (PRJNA678740) [50], photoperiod (PRJNA475903) [51], downy mildew (PRJNA285071) [52], powdery mildew (PRJNA321023) [53], root-knot nematode stress (PRJNA419665) [54], blight (PRJNA472169) [55], angular leaf spot (PRJNA704621) [56], necrotic ringspot virus (PRJNA837466), oomycete pathogen (PRJNA345040) [57], and cucumber green mottle mosaic virus (PRJNA646644) [58] were retrieved from NCBI database. Transcriptome analysis was then re-conducted using the genomic data from the ChineseLong_V3 version of cucumber. Expression heatmap of Lhc family across various cucumber tissues and organs was drawn using TBtools software (version 2.0).

2.6. qRT-PCR Analysis of Lhc Family in Cucumber Under High Temperature and Salt Stress

Cucumber seeds of varieties ‘9930’ provided by Nanjing Agricultural University were germinated and grown in pots containing aseptic organic substrate under controlled environmental conditions: 26 °C/20 °C (day/night) temperatures, 16 h·d−1 photoperiod, light flux density of 360 µmol·m−2·s−1, and 70% relative humidity. At the two-leaf stage, seedlings were carefully removed from the substrate, and roots were rinsed before transfer to hydroponic tanks containing Hogan’s nutrient solution. After one day of acclimation in hydroponics, plants were subjected to either heat stress (42 °C) or salt stress (75 mM NaCl), with untreated plants serving as controls. For heat stress, the third true leaves were sampled at 0, 3, and 6 h post-treatment. For salt stress, sampling occurred at 0 and 6 h after treatment. All leaf samples were immediately frozen in liquid nitrogen and stored in 50 mL tubes at −80 °C until analysis. Three biological replicates were collected for each treatment/time point combination. Total RNA of frozen leaves was extracted using TRIzol RNA Extraction Reagent (ATG Biotechnology) following the manufacturer’s protocol. First-strand cDNA was synthesized using HiScript III RT SuperMix for qPCR (Vazyme). Candidate Lhc genes showing significant differential expression in transcriptome analyses under salt or heat stress were selected for validation. Primer sequences (Table S1) were designed using Primer 5 software (Premier Biosoft) and synthesized by Shanghai Bioengineering Company. qRT-PCR analyses were performed on a CFX96 Touch Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA), following the method of ChamQ Universal SYBR qPCR Master Mix (Vazyme). CsActin was used as an internal reference gene. Relative gene expression levels were calculated using the 2−ΔΔCt method. All data were analyzed by one-way ANOVA using GraphRad Prism8 software (GraphPad), with significant differences between treatments determined by Duncan’s multiple range test.

3. Results

3.1. Basic Information of Lhc Family Members in Cucumber

Based on the published genomic information of cucumber ChineseLong_V3, a total of 21 Lhc family members were identified from the cucumber genome using bioinformatics methods. The gene CDS ranged from 579 to 867 bp, the number of coding amino acids ranged from 192 to 288, molecular weights ranged from 8.70 to 73.82 kD, and fat coefficients ranged from 64.37 to 100.63 (Table 1). The theoretical isoelectric points of 23 Lhc proteins ranged from 20.21 to 31.09. Except for CsLhca1, CsLhca4, CsLhca4.1, CsLhca4.2, CsLhca5, and CsLhca6, which are unstable proteins (instability index > 40), instability indices of the remaining Lhcs were less than 40, being stable proteins (Table 1). Among 21 Lhcs, the average hydrophilicity of 13 Lhc proteins was less than zero, indicating they are hydrophilic, while the average hydrophilicity of 8 Lhc proteins was greater than zero, indicating they are hydrophobic (Table 1). Predictions results of subcellular localization indicated that cucumber Lhc proteins were primarily localized in cell membrane and chloroplasts (Table 1).

3.2. Chromosomal Localization of Lhc Family in Cucumber

The distribution of the 21 Lhc family members on cucumber chromosomes was mapped based on chromosomal localization. Each cucumber chromosome harbors Lhc family members, with Chr. 1 containing the most, having six Lhc genes; of these, the CsLhca4/CsLhca4.1 are segmental duplicates, and CsLhcb6 and CsLhcb6.1 on Chr. 6 are also segmental duplicates. Chr. 2 and 7 had the fewest Lhcs, with only one Lhc each. Chr. 3 and 5 each contained three Lhsc, Chr. 4 had two Lhcs, and Chr. 6 contained five Lhcs, with CsLhc1.4 and CsLhc1.5 as tandem duplicates (Figure 1).

3.3. Clustering Analysis of Lhc Family in Cucumber, Arabidopsis, and Rice

To clarify the affinities and biological functions among the members of the CsLhc family, multiple sequence alignment analyses were conducted for the identified Lhc family in C. sativus, O. sativa and the model plant A. thaliana, and phylogenetic trees were constructed (Figure 2). Based on the classification of the Arabidopsis Lhc family, the CsLhc family was split into five subfamilies: Lhca, Lhcb, CP24, CP26, and CP29. Among these, the Lhca subfamily contained the most genes, with nine, while the CP26 and CP29 subfamilies had the fewest, with one gene each. The Lhcb subfamily contained eight genes, and the CP24 subfamily contained two genes. Four pairs of direct homologs between the CsLhc family and AtLhc family were identified: CsLhca6/AtLhca6, CsLhca1/AtLhca1, CsLhca4/AtLhca4, and CsLhcb7/AtLhcb7. Four pairs of direct homologs between the CsLhc family and OsLhc family were also identified: CsLhca3/OsLhca3, CsLhca5/OsLhca5, CsLhcb3/OsLhcb3, and CsLhcb5/OsLhcb5. Four pairs of paralogous homologs existed within the cucumber Lhc family: CsLhca4.2/CsLhca4.3, CsLhcb1.2/CsLhcb1.3, CsLhcb1.4/CsLhcb1.5, and CsLhcb6/CsLhcb6.1. Lhc genes that are relatively similar in evolutionary relationships tend to be structurally and functionally similar. Therefore, on the basis of similar genes in the model plant Arabidopsis, the biological functions of Lhc in cucumber could be hypothesized.

3.4. Structure and Conserved Sequence Analysis of Lhc Family in Cucumber

Using TBtools 2.0 software to draw a gene clustering map and a structural schematic diagram, we found that the CsLhc family contains five subfamilies, i.e., Lhca, Lhcb, CP24, CP26, and CP29 (Figure 3), which is consistent with the Lhc clustering results in cucumber and Arabidopsis (Figure 2). Conserved amino acid sequences of Lhc in cucumber were analyzed using online software MEME 5.5.8 software (Table 2). The schematic structure of CsLhc revealed that conserved amino acid sequences of Lhc proteins in different subfamilies were different, while Lhc proteins in the same subfamily had the same conserved sequences; e.g., in the Lhca subfamily, most of the proteins contained motifs 3, 1, 6, 1, and 4 with the same arrangement order, whereas most of the proteins in the Lhcb subfamily contained motifs 8, 7, 3, 1, 2, 10, 5, 1, 4, 9, and had the same sequences, which suggests that the different distribution of motifs in different Lhc subfamilies led to their possible functional diversity in the evolutionary process; meanwhile, the similar conserved motifs of Lhc in the same subfamily showed similar functions.

3.5. Collinearity Analysis of Lhc Family in Cucumber, Arabidopsis, and Rice

Collinearity analysis of the Lhc families in three crops revealed that six Lhcs in cucumber (CsLhca4, CsLhca4.1, CsLhca6, CsLhcb1.4, CsLhcb2, CsLhcb4) and seven Lhcs in A. thaliana (AtLhca4, AtLhca6, AtLhcb1.2, AtLhcb1.5, AtLhcb2.2, AtLhcb4.1, AtLhcb4.3) formed eight linear relationships. One Lhc, CsLhca6, in cucumber and one Lhc, OsLhca6, in rice exhibited a linear relationship. Additionally, 15 Lhcs in cucumber (CsLhca1, CsLhca2, CsLhca3, CsLhca4.2, CsLhca4.3, CsLhca5, CsLhcb1.1, CsLhcb1.2, CsLhcb1.3, CsLhcb1.5, CsLhcb3, CsLhcb5, CsLhcb6, CsLhcb6.1, CsLhcb7) were more conserved and did not exhibit covariation with Lhc in Arabidopsis and rice. This suggests that the Lhc family in cucumber and Arabidopsis are evolutionarily more homologous (Figure 4).

3.6. Cis Elements Analysis of the Promoter Sequence of Cucumber Lhc Family

Fourteen major cis-elements were identified in the promoter of the cucumber Lhc family (Figure 5). Among these, cis-elements’ responses to light were the most numerous class, including ACE, Box4, G-box, and I-box, which together accounted for 55% of total cis-elements. Additionally, other cis-elements related to hormone (auxin, gibberellin, salicylic acid, abscisic acid, MeJA) responses, stress responses (drought, low temperature), endosperm expression, and meristematic tissue expression were also identified. The promoters of different Lhcs contained distinct cis-elements, suggesting that the Lhc family plays diverse roles in the cucumber life process.

3.7. Tissue-Specific Expression Analysis of Lhc Family in Cucumber

A transcriptome sequencing analysis was conducted to generate a heat map of Lhc family gene expression across different tissues (Figure 6). We found that eight Lhcs, including CsLhca6, CsLhca4.1, CsLhca5, CsLhcb7, CsLhca4.2, CsLhca4.3, CsLhcb1.4, and CsLhcb1.1, were not expressed in roots, while the remaining genes were expressed in roots. In stems, only CsLhca6 was not expressed, while the remaining genes were expressed, with most showing high expression. In male flowers, CsLhca6, CsLhca4.1, CsLhca5, CsLhcb6, and CsLhcb6.1 were not expressed, while the remaining genes were expressed, with CsLhca4.2 showing the highest expression. All Lhc genes in female flowers and leaves were expressed, with some showing higher expression levels. CsLhca6 was not expressed in the ovary, unfertilized ovary, and fertilized ovary, while the remaining genes were expressed, with most showing higher expression levels. In tendrils and basal tendrils, both CsLhca6 and CsLhca4.1 were not expressed, while the remaining genes were expressed, with most showing higher expression levels.

3.8. Expression Pattern Analysis of Cucumber Lhc Family Under Abiotic Stress

We re-analyzed transcriptome sequencing data with the cucumber genome and generated various abiotic stresses’ response expression heat maps of the Lhc family (Figure 7). CsLhca1-4, CsLhca4.1-4.3, CsLhcb1.1-1.5, CsLhcb2, CsLhcb3, CsLhcb6, and CsLhcb6.1 exhibited significantly down-regulated expression under high-temperature stress compared to the control (Figure 7A); the other gene expression did not differ from the control. Treated by low temperature, only CsLhca4 exhibited significant down-regulation of expression (Figure 7B); the other gene expression had no difference from the control. Under salt stress, only CsLhcb7 exhibited significant down-regulation, while CsLhca4.2 was significantly up-regulated (Figure 7C). Under waterlogging stress, CsLhca4.3 and CsLhcb1.3 exhibited significant down-regulation of expression, while CsLhcb5 was up-regulated compared to the control (Figure 7D). Under photoperiod treatment, CsLhca1, CsLhca4.2, CsLhcb6, and CsLhcb6.1 exhibited significant down-regulation of expression, while CsLhcb1.1, CsLhcb1.2, CsLhcb1.5, and CsLhcb2 were significantly up-regulated compared to the control (Figure 7E).

3.9. Expression Pattern Analysis of Cucumber Lhc Family Under Biotic Stress

Transcriptome sequence drawing support from the cucumber genome was re-analyzed and generated various biotic stresses’ response expression heat maps of the Lhc family (Figure 8). Under downy mildew stress treatment, the expression levels of four Lhcs (CsLhca1, CsLhca3, CsLhcb2, CsLhcb4) in susceptible material were significantly down-regulated on the first day of inoculation, after which they began to up-regulate relative to the control. CsLhca2, CsLhca4, CsLhca4.1, CsLhca4.3, CsLhcb1.1-1.5, CsLhcb3, CsLhcb5, CsLhcb6, and CsLhcb6.1 began to significantly up-regulate expression after the second day of inoculation in disease-sensitive materials (Figure 8A). Under powdery mildew stress treatment, only the CsLhcb1.5 exhibited significant up-regulation in the susceptible material, whereas the CsLhca3 showed significant down-regulation in the resistant plants (Figure 8B). After root-knot nematode inoculation, CsLhca1-4, CsLhca4.1, CsLhca4.3, CsLhcb1.1-1.5, CsLhcb2-4, CsLhcb4.2, CsLhcb5, CsLhcb6, and CsLhcb6.1 were significantly up-regulated in both resistant and susceptible plants, while CsLhcb7 exhibited significant up-regulation only in susceptible plants, with up-regulation in resistant materials being non-significant (Figure 8C). Under blight stress treatment, all genes initially up-regulated expression then began to down-regulate after 96 h of treatment (Figure 8D). Under angular leaf spot treatment, Lhc in both resistant and susceptible materials showed a tendency to down-regulate expression (Figure 8E). Under necrotic ringspot virus treatment, five genes, including CsLhca1, CsLhca4.3, CsLhca5, CsLhcb1.1, and CsLhcb6, underwent significant down-regulation of expression (Figure 8F). Under oomycete pathogen treatment, 19 Lhc, including CsLhca1-4, CsLhca4.1, CsLhca4.3, CsLhca5, CsLhca6, CsLhcb1.1-1.5, CsLhcb2-6, and CsLhcb6.1, down-regulated significantly in both resistant and susceptible materials, while CsLhca4.2 was up-regulated in susceptible materials and down-regulated in resistant materials (Figure 8G). Under cucumber green mottle mosaic virus treatment, the expression of 20 Lhcs, including CsLhca1-6, CsLhca4.1-4.3, CsLhca5-6, CsLhcb1.1-1.5, CsLhcb2-6, and CsLhcb6.1, down-regulated significantly (Figure 8H). These findings suggest that Lhc in cucumber plays a significant role in coping with various biotic stresses.

3.10. Regulatory Patterns Analysis of Cucumber Lhc in Response to Abiotic and Biotic Stresses

By analyzing the expression patterns of the cucumber Lhc family under abiotic and biotic stresses, as described above, we generated a heatmap of differentially expressed Lhc (Figure 9). It was evident that all the Lhc family was involved in at least one stress. Some cucumber Lhcs, such as CsLhcb7, exhibit differential expression under individual abiotic and biotic stresses, though their expression patterns differ. Additionally, some cucumber Lhcs, such as CsLhca5 and CsLhca6, are only involved in biotic stress and exhibit down-regulation of expression. The regulatory patterns of the cucumber Lhc family in response to abiotic and biotic stresses will inform subsequent in-depth studies on the molecular biology of these genes.

3.11. Expression Profiles of Cucumber Lhc Family in Response Low Temperature and Salt Stress

As shown in Figure 10, the expression levels of all four detected genes were significantly downregulated under 42 °C heat stress compared to the control (p < 0.0001), consistent with the transcriptome data. Notably, the expression of CsLhcb1.1, CsLhcb1.2, and CsLhca3 exhibited highly significant differences between 3 h and 6 h high-temperature treatments (p < 0.0001). Similarly, the expression of CsLhcb1.4 also showed significant variation between these time points, though at a slightly lower significance threshold (p < 0.01).
Under 75 mM NaCl treatment for 6 h, CsLhcb1.1 showed a significant downregulation (p < 0.0001), which contrasted with the transcriptome data. In contrast, the other three genes all displayed up-regulated expression under salt stress, consistent with the transcriptome results. Compared to the control, CsLhcb1.2 and CsLhca4.2 were up-regulated with an extremely significant increase (p < 0.0001) and CsLhcb1.4 was highly significant in an increase in expression (p < 0.01) after 6 h of salt treatment (Figure 11).

4. Discussion

In recent years, with the continued advancement and widespread application of genome sequencing technology, an increasing amount of plant genome sequence data has been published [59], leading to the identification of many important gene families. As antenna proteins, LHC superfamily proteins play critical roles in light energy capture and transfer, and the response to adversities. The Lhc families in the model plants A. thaliana and rice were first identified [14], and later reported in other plants, including cotton [9] and cassava [15]. Currently, a high-quality cucumber genome is available, but identification and functional studies of the cucumber Lhc family have not been yet conducted, which limits the exploration of their biological functions. Therefore, in this study, the genomic information of the C. sativus ChineseLong_V3 genome was used to identify the Lhc family and transcriptome data were re-analyzed to investigate the expression patterns of these genes in different tissues and organs, as well as their response to various adversities. This research could provide a reference for in-depth studies on the function of Lhc and offer a theoretical basis for the molecular breeding of stress-resistant cucumber varieties.
The number of the Lhc family varies among different plant species. A total of 21 Lhc family members in cucumber were identified, while the number of Lhcs identified in A. thaliana, rice [14], cassava [15], cotton [9], castor [16], and apple [3] were 34, 29, 35, 55, 28, and 27, respectively. A total of 21 Lhcs in cucumber were classified into five subfamilies, which was identical with the phylogenetic analysis of Lhc in A. thaliana and rice [14]. However, the gene structures among subfamily members had significant differences, although the motifs and structures within each subfamily were largely conserved. The CsLhc proteins are mainly located in chloroplasts and the Lhc genes in the subfamilies have the same motif and structures, suggesting that family members are vital for photosynthesis, which is in line with the results of previous studies [60]. In this research, four pairs of direct homologous genes were found between cucumber, and Arabidopsis, based on phylogenetic analysis, there were also four pairs of direct homologous genesand between cucumber and rice. In general, homologous genes often tend to preserve similar functions throughout the evolution of distinct species. Hence, we speculated that four pairs of Lhc genes likely possess analogous biological roles. We found that six Lhc genes in cucumber were collinear with AtLhc, while only one CsLhc was collinear with OsLhc, suggesting that the Lhc family in cucumber is more closely related to those in A. thaliana than to those in rice. Gene family expansion in plants was mainly caused by fragmentation duplication and tandem duplication [61]. Only one tandem repeat gene and two segmental repeat gene pairs were identified in the cucumber Lhc family, making clear that Lhc expansion in cucumber is primarily due to segmental duplications.
Cis-elements in the promoters can regulate gene expression, so the nature of cis-elements determined the function of genes. In this study, 14 kinds of cis-elements were predicted to exist in the promoter regions of the CsLhc genes, among which, light responsiveness elements are the most abundant ones, which is consistent with the results of previous studies [60]. Cis-elements related to hormone responses, stress responses, endosperm expression, and meristematic tissue expression were also identified. These studies suggest that CsLhc genes play a crucial role not only in the process of light-regulated growth and development but also in regulating plant resistance to various stresses [18,19,20,60]. We found that some genes of the 21 in the Lhc family were expressed in all cucumber tissues, but expression levels were lower in roots and staminate flowers, while higher expression levels were observed in the stem, pistillate flower, ovary, leaf, tendrils, and basal tendrils. The idiosyncrasy of the Lhc family expression varied across different tissues, where they likely collaboratively regulated cucumber growth and development.
Our results indicate that at least one CsLhc gene was expressed under all stress conditions, although CsLhca5 and CsLhca6 had no expression under abiotic stresses. One Lhc was expressed under both low-temperature and salt stresses, while other genes were not, which is consistent with previous studies where high salt and low temperature reduced the expression of most Lhca and Lhcb genes in Zostera marina [62]. Our qPCR validation showed that high temperature reduced the down-expression of CsLhcb1.1, CsLhcb1.2, CsLhcb1.4, and CsLhca3, and the expression of CsLhcb1.1 declined under NaCl treatment too, while CsLhcb1.2, CsLhcb1.4 and CsLhca4.2 showed upward changes under NaCl treatment. Analysis of CsLhc family expression in response to biotic stresses revealed a few genes expressed in response to powdery mildew and necrotic ringspot virus, while most Lhcs were expressed in other biotic stress conditions; in particular under downy mildew, root-knot nematode stresses, and blight stress, the most Lhc genes were up-regulated, suggesting they were involved in anti-adversity responses. These high up-regulated Lhc genes could be cloned and transformed for genetic improvement in the future. But the function validation and utilization of the Lhc gene family in cucumber have not been carried out and need further research.
Nitrogen is the main component of RuBisCO and photosynthetic pigments, participating in the process of photosynthesis. In comparative transcriptome analysis of tea plants (Camellia sinensis), all LHC proteins were significantly higher in TC12 (high nitrogen use efficiency genotype) than LJCY (low nitrogen use efficiency genotype), which suggests that TC12 might enhance photosynthesis by up-regulating the expression of LHC [13]. The expression of LHC in Cenchrus alopecuroides is up-regulated by exogenous melatonin induction to alleviate water deficit [63].

5. Conclusions

We identified 21 Lhc genes in the cucumber genome. Our analysis revealed that the Lhc family are distributed across seven chromosomes. The cucumber Lhc family was divided into five subfamilies, with each subfamily exhibiting highly conserved gene sequences. However, gene structures and protein domains varied among subfamilies. The promoter regions of the CsLhc genes contained a significant number of cis-elements, indicating that the expression of CsLhc genes was controlled by a complex regulatory network. The expression style of the cucumber Lhc family was tissue-specific and stress-specific, suggesting that it may collaboratively regulate cucumber growth and development in response to various environmental conditions. CsLhcb1.1, CsLhcb1.2, CsLhcb1.4, CsLhca3, and CsLhca4.2 were confirmed to appear to have expression change using qPCR analysis under high temperature and salt stress. This study provides a valuable reference for deep study of the biological functions of cucumber Lhc and potential candidates for molecular breeding of cucumber resistance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11070736/s1, Table S1: RT-qPCR primers for expression analysis of CsLhc family genes.

Author Contributions

Conceptualization, Y.M. and K.Z.; methodology, Y.M.; software, K.Z.; formal analysis, Y.M. and K.Z.; data curation, Y.M. and K.Z.; writing—original draft preparation, K.Z.; writing—review and editing, Y.M.; visualization, K.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research and APC were funded by Stable Talent Project from Anhui Science and Technology University.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Acknowledgments

Thanks to Yu Ning for the revision and polishing of the English language of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The distribution of Lhc family on cucumber chromosomes. Note: Chromosomes are indicated with long grey bars, and chromosome numbers are shown at the left of each chromosome. Genes marked in blue are tandem duplication gene pairs, and genes marked in red are segmental duplication gene pairs.
Figure 1. The distribution of Lhc family on cucumber chromosomes. Note: Chromosomes are indicated with long grey bars, and chromosome numbers are shown at the left of each chromosome. Genes marked in blue are tandem duplication gene pairs, and genes marked in red are segmental duplication gene pairs.
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Figure 2. Phylogenetic analysis of Lhc from Cucumis sativus (Cs, red gene name and pentagram), Oryza sativa (Os, blue gene name and triangle), and A. thaliana (At, green gene name and circle).
Figure 2. Phylogenetic analysis of Lhc from Cucumis sativus (Cs, red gene name and pentagram), Oryza sativa (Os, blue gene name and triangle), and A. thaliana (At, green gene name and circle).
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Figure 3. Exon–intron structures of Lhc and a schematic diagram of Lhc proteins’ amino acid motifs.
Figure 3. Exon–intron structures of Lhc and a schematic diagram of Lhc proteins’ amino acid motifs.
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Figure 4. Collinearity relationships of Lhc family in cucumber, Arabidopsis, and rice. Note: green color line indicates the colinearity of Lhc genes between O. sativa and C. sativus, blue color line indicates the colinearity of Lhc genes between A. thaliana and C. sativus.
Figure 4. Collinearity relationships of Lhc family in cucumber, Arabidopsis, and rice. Note: green color line indicates the colinearity of Lhc genes between O. sativa and C. sativus, blue color line indicates the colinearity of Lhc genes between A. thaliana and C. sativus.
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Figure 5. Cis-elements analysis of the promoters of Lhc family. (A): Numbers of cis-elements. (B): Relative proportions of different response cis-elements.
Figure 5. Cis-elements analysis of the promoters of Lhc family. (A): Numbers of cis-elements. (B): Relative proportions of different response cis-elements.
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Figure 6. The expression heatmap of Lhc family in different tissues. Note: The data in the boxes indicate original FPKM values.
Figure 6. The expression heatmap of Lhc family in different tissues. Note: The data in the boxes indicate original FPKM values.
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Figure 7. The expression heatmaps of cucumber Lhc family under abiotic stress treatments. (A): The expression patterns under high-temperature stress. CT, HT_3h and HT_6h: high-temperature treatment for 0 h, 3 h and 6 h, respectively. (B): The expression patterns under low-temperature stress. CT, CS_2h, CS_6h, and CS_12h: low-temperature treatment for 0 h, 2 h, 6 h, and 12 h, respectively. (C): The expression patterns under salt stresses. CT: control; Salt: salt stress. (D): The expression patterns under waterlogging stress. S: sensitive plant; T: tolerance plant; Ctrl: untreated plants cultivated under optimal conditions; 1xH: non-primed plants waterlogged for 7 days only once; Rec: plants after 7 days of waterlogging and 14 days of recovery; 2xH: primed plants waterlogged for 7 days and after 14 days of recovery, then waterlogged again. (E): The expression patterns under photoperiod stress. LD1: long-day treatment for 7, 14, and 21 days; LD2: long-day treatment for 37 and 44 days; SD1: short-day treatment for 7, 14, and 21 days; SD2: short-day treatment for 37 and 44 days. The data in the grids represent original FPKM values.
Figure 7. The expression heatmaps of cucumber Lhc family under abiotic stress treatments. (A): The expression patterns under high-temperature stress. CT, HT_3h and HT_6h: high-temperature treatment for 0 h, 3 h and 6 h, respectively. (B): The expression patterns under low-temperature stress. CT, CS_2h, CS_6h, and CS_12h: low-temperature treatment for 0 h, 2 h, 6 h, and 12 h, respectively. (C): The expression patterns under salt stresses. CT: control; Salt: salt stress. (D): The expression patterns under waterlogging stress. S: sensitive plant; T: tolerance plant; Ctrl: untreated plants cultivated under optimal conditions; 1xH: non-primed plants waterlogged for 7 days only once; Rec: plants after 7 days of waterlogging and 14 days of recovery; 2xH: primed plants waterlogged for 7 days and after 14 days of recovery, then waterlogged again. (E): The expression patterns under photoperiod stress. LD1: long-day treatment for 7, 14, and 21 days; LD2: long-day treatment for 37 and 44 days; SD1: short-day treatment for 7, 14, and 21 days; SD2: short-day treatment for 37 and 44 days. The data in the grids represent original FPKM values.
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Figure 8. The expression heatmaps of cucumber Lhc family under biotic stress. (A): The expression patterns under downy mildew stress; S: susceptible plants; R: resistant plants; 1 dpi, 2 dpi, 3 dpi, 4 dpi, and 6 dpi were respectively 1, 2, 3, 4, and 6 days post-inoculation. (B): The expression patterns under powdery mildew stress; S: susceptible plants; R: resistant plants; CT: control; 48 hpi: 48 h post-inoculation. (C): The expression patterns under root-knot nematode stress; S: susceptible plants; R: resistant plants; CT: control; 1 dpi, 2 dpi, and 3 dpi were respectively 1, 2, and 3 days post-inoculation. (D): The expression patterns under blight stress; CT: control; Foc: Fusarium oxysporum f. sp. Cucumerinum; 24 hpi, 48 hpi, 96 hpi, and 192 hpi were respectively 24, 48, 96, 192 h post-inoculation. (E): The expression patterns under angular leaf spot stress; CT: control; Gy14: resistant cucumber line; B10: susceptible cucumber line; 1 dpi, 3 dpi were 1, 3 days post-inoculation, respectively. (F): The expression patterns under necrotic ringspot virus stress; MOCK: non-inoculation; PNRSV: inoculation with necrotic ringspot virus. (G): The expression patterns under oomycete pathogen stress; S: susceptible plants; R: resistant plants; 8 dpp, 16 dpp were 8, 16 days post-pollination, respectively. (H): The expression patterns under cucumber green mottle mosaic virus stress; CT-3d and CT-20d were control for No 3 and 20 days; 3 dpi and 20 dpi were 3 and 20 days post-inoculation. The data in the grids represent original FPKM values.
Figure 8. The expression heatmaps of cucumber Lhc family under biotic stress. (A): The expression patterns under downy mildew stress; S: susceptible plants; R: resistant plants; 1 dpi, 2 dpi, 3 dpi, 4 dpi, and 6 dpi were respectively 1, 2, 3, 4, and 6 days post-inoculation. (B): The expression patterns under powdery mildew stress; S: susceptible plants; R: resistant plants; CT: control; 48 hpi: 48 h post-inoculation. (C): The expression patterns under root-knot nematode stress; S: susceptible plants; R: resistant plants; CT: control; 1 dpi, 2 dpi, and 3 dpi were respectively 1, 2, and 3 days post-inoculation. (D): The expression patterns under blight stress; CT: control; Foc: Fusarium oxysporum f. sp. Cucumerinum; 24 hpi, 48 hpi, 96 hpi, and 192 hpi were respectively 24, 48, 96, 192 h post-inoculation. (E): The expression patterns under angular leaf spot stress; CT: control; Gy14: resistant cucumber line; B10: susceptible cucumber line; 1 dpi, 3 dpi were 1, 3 days post-inoculation, respectively. (F): The expression patterns under necrotic ringspot virus stress; MOCK: non-inoculation; PNRSV: inoculation with necrotic ringspot virus. (G): The expression patterns under oomycete pathogen stress; S: susceptible plants; R: resistant plants; 8 dpp, 16 dpp were 8, 16 days post-pollination, respectively. (H): The expression patterns under cucumber green mottle mosaic virus stress; CT-3d and CT-20d were control for No 3 and 20 days; 3 dpi and 20 dpi were 3 and 20 days post-inoculation. The data in the grids represent original FPKM values.
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Horticulturae 11 00736 g009
Figure 9. The expression heatmap of cucumber Lhc family under stress. Note: Gray represents no change in expression level, red represents up-regulation, green represents down-regulation, and blue represents both up-regulation and down-regulation.
Figure 9. The expression heatmap of cucumber Lhc family under stress. Note: Gray represents no change in expression level, red represents up-regulation, green represents down-regulation, and blue represents both up-regulation and down-regulation.
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Horticulturae 11 00736 g010
Figure 10. The expression of cucumber Lhc family members under 42 °C temperature. Note: ** represents significant differences (p < 0.01), **** represents significant differences (p < 0.0001), among different treat times.
Figure 10. The expression of cucumber Lhc family members under 42 °C temperature. Note: ** represents significant differences (p < 0.01), **** represents significant differences (p < 0.0001), among different treat times.
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Horticulturae 11 00736 g011
Figure 11. The expression of cucumber Lhc family members under salt stress. Note: ** represents significant differences (p < 0.01), **** represents significant differences (p < 0.0001), among different treat times.
Figure 11. The expression of cucumber Lhc family members under salt stress. Note: ** represents significant differences (p < 0.01), **** represents significant differences (p < 0.0001), among different treat times.
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Table 1. The physicochemical characteristics of 23 members in CsLhc family.
Table 1. The physicochemical characteristics of 23 members in CsLhc family.
Family MemberGene IDCDS SizeNumber of Amino AcidsMolecular WeightpIInstability IndexAliphatic IndexGrand Average of HydropathicityPrediction of Subcellular Location
CsLhca1CsaV3_5G025740.168122624.436.7442.3987.210.034chloroplast
CsLhca2CsaV3_5G030270.181627129.096.4234.7684.020.050chloroplast
CsLhca3CsaV3_1G043710.182227329.428.6729.6680.48−0.063chloroplast
CsLhca4CsaV3_1G002410.175925227.676.9049.8284.01−0.08chloroplast
CsLhca4.1CsaV3_1G005600.175325027.796.4341.1581.92−0.182chloroplast
CsLhca4.2CsaV3_3G012890.157919220.219.5644.8488.960.143cell membrane
CsLhca4.3CsaV3_6G034480.181927229.056.7624.70102.210.272chloroplast
CsLhca5CsaV3_2G027790.178025928.487.1740.5792.66−0.022cell membrane
CsLhca6CsaV3_1G041230.180426729.355.8643.0477.53−0.131cell membrane
CsLhcb1.1CsaV3_1G032510.179826528.275.2926.8978.08−0.036chloroplast
CsLhcb1.2CsaV3_3G031580.179826528.315.4725.4378.83−0.045chloroplast
CsLhcb1.3CsaV3_6G013800.180426728.355.1525.6779.36−0.024chloroplast
CsLhcb1.4CsaV3_6G051520.179826528.195.1425.5378.49−0.033chloroplast
CsLhcb1.5CsaV3_6G051530.179826528.145.1425.5378.87−0.013chloroplast
CsLhcb2CsaV3_7G002620.179826528.545.4628.7179.58−0.036chloroplast
CsLhcb3CsaV3_3G005950.179526428.544.8920.7386.140.011chloroplast
CsLhcb4CsaV3_4G026100.185528431.095.6737.7188.66−0.082chloroplast
CsLhcb5CsaV3_5G039350.186728830.935.3332.4288.19−0.016chloroplast
CsLhcb6CsaV3_1G019610.161820521.9610.7034.9288.540.001cell membrane
CsLhcb6.1CsaV3_6G005340.176825527.226.7523.2486.940.089chloroplast
CsLhcb7CsaV3_4G037740.181627130.104.8635.5696.490.086cell membrane
Table 2. The motifs information of Lhc proteins in cucumber.
Table 2. The motifs information of Lhc proteins in cucumber.
MotifSequenceNumber of Amino AcidsPfam Annotation
motif 1EJKNGRLAMLAMLGFFVPEILT22Chlorophyll a/b binding protein
motif 2RNGVKFGEAVWFKAGSQIFSEGGLDYLGNPSLVHAQSILAIWACQVVLMGAVEGYRIAG59Chlorophyll a/b binding protein
motif 3PSYLDGELPGDYGFDPLGLSADPE24Chlorophyll a/b binding protein
motif 4GKGPLENLADHLADPVHNNAW21-
motif 5GSFDPLGLADDPEAFAELKVK21-
motif 6IGIINVPSWYDAGKAEYFADSSTLFVIEFILFGWVEGRRWQDIKNPGSVNQDPIFPQYKLPPNDVGYPGG70Chlorophyll a/b binding protein
motif 7SGSPWYGPDRVKYLGPFSGEP21-
motif 8AASSMALSSPSFAGQAVKLSPTAPEJQGNAKFTMRKTASKS41-
motif 9AYATNFVPGK10-
motif 10PLGEVTDPIYP11-
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Miao, Y.; Zhang, K. Genome-Wide Identification of Cucumber Lhc Genes’ Family and Their Expression Analysis. Horticulturae 2025, 11, 736. https://doi.org/10.3390/horticulturae11070736

AMA Style

Miao Y, Zhang K. Genome-Wide Identification of Cucumber Lhc Genes’ Family and Their Expression Analysis. Horticulturae. 2025; 11(7):736. https://doi.org/10.3390/horticulturae11070736

Chicago/Turabian Style

Miao, Yongmei, and Kaijing Zhang. 2025. "Genome-Wide Identification of Cucumber Lhc Genes’ Family and Their Expression Analysis" Horticulturae 11, no. 7: 736. https://doi.org/10.3390/horticulturae11070736

APA Style

Miao, Y., & Zhang, K. (2025). Genome-Wide Identification of Cucumber Lhc Genes’ Family and Their Expression Analysis. Horticulturae, 11(7), 736. https://doi.org/10.3390/horticulturae11070736

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