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Article

Identification and Molecular Characterization of the CAMTA Gene Family in Solanaceae with a Focus on the Expression Analysis of Eggplant Genes under Cold Stress

by
Peng Cai
1,2,†,
Yanhong Lan
1,2,†,
Fangyi Gong
1,2,
Chun Li
1,2,
Feng Xia
1,2,
Yifan Li
1,2 and
Chao Fang
1,2,*
1
Horticulture Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China
2
Vegetable Germplasm Innovation and Variety Improvement Key Laboratory of Sichuan Province, Chengdu 610066, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2024, 25(4), 2064; https://doi.org/10.3390/ijms25042064
Submission received: 22 December 2023 / Revised: 3 February 2024 / Accepted: 6 February 2024 / Published: 8 February 2024
(This article belongs to the Section Molecular Plant Sciences)
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Versions Notes

Abstract

:
Calmodulin-binding transcription activator (CAMTA) is an important calmodulin-binding protein with a conserved structure in eukaryotes which is widely involved in plant stress response, growth and development, hormone signal transduction, and other biological processes. Although CAMTA genes have been identified and characterized in many plant species, a systematic and comprehensive analysis of CAMTA genes in the Solanaceae genome is performed for the first time in this study. A total of 28 CAMTA genes were identified using bioinformatics tools, and the biochemical/physicochemical properties of these proteins were investigated. CAMTA genes were categorized into three major groups according to phylogenetic analysis. Tissue-expression profiles indicated divergent spatiotemporal expression patterns of SmCAMTAs. Furthermore, transcriptome analysis of SmCAMTA genes showed that exposure to cold induced differential expression of many eggplant CAMTA genes. Yeast two-hybrid and bimolecular fluorescent complementary assays suggested an interaction between SmCAMTA2 and SmERF1, promoting the transcription of the cold key factor SmCBF2, which may be an important mechanism for plant cold resistance. In summary, our results provide essential information for further functional research on Solanaceae family genes, and possibly other plant families, in the determination of the development of plants.

1. Introduction

The Solanaceae family, comprising approximately 3000 plant species, plays a crucial role in the realm of vegetable crops and holds economic significance surpassed only by cereals and leguminous plants. Notable members of this family include potatoes, tomatoes, chili peppers, eggplants, goji berries, tobacco, and Bidong eggplants. Fruits from the Solanaceae family boast a wealth of nutrients, encompassing proteins, fats, carbohydrates, vitamins, antioxidant polyphenols, and essential trace elements like calcium, phosphorus, and iron. This nutritional richness makes them highly sought after as a popular food source and a vital vegetable crop [1,2]. The global cultivation of Solanaceae crops is extensive, particularly in Asia, with China leading as the largest producer. In 2020, China contributed to 68% of the global production. However, the cultivation of Solanaceae crops faces challenges, and their yield and quality are often compromised by cold damage [3,4]. Therefore, investigating the mechanisms through which crops respond to cold damage has become an urgent necessity. With continuous progress in genomics and bioinformatics, the isolation and cloning of and functional research on the cold regulatory factor CAMTA (Calmodulin-binding transcription activator) have gained considerable attention.
CAMTA represents an essential class of structurally conserved calmodulin-binding proteins which are widely present in eukaryotes [5,6,7]. The CAMTA protein family’s secondary structures consistently feature a conserved functional domain module. From the N-end, these structures comprise a CG-1 DNA binding domain, a transcription factor immunoglobulin (TIG) domain, an ankyrin repeat (ANK) domain, a Ca2+- dependent calmodulin binding domain (CaMBD), and a tandem repeat IQ motif (IQXXXRGXXX) functional domains [8,9]. Furthermore, evidence suggests that the TIG domain associates with non-specific DNA contact with transcription factors, the ANK repeat is linked to protein interactions, and the IQ motif can bind to calmodulin (CaM) or calmodulin-like (CML) proteins without relying on Ca2+ [10,11]. However, recent research has unveiled that the CG-1 domain of Arabidopsis CAMTA3 imparts temperature dependence to the ability of CAMTA3 TAD (newly identified transcriptional activation domain) to induce gene expression [12].
The CAMTA family has been identified in various plant species, including Arabidopsis thaliana [13], Triticum aestivum [14], Musa nana [15], Oryza sativa [16], Brassica napus [17], and Camellia sinensis [18]. CAMTA is a relatively small, plant-specific family usually consisting of fewer than 10 members, although there were 24 members identified in B. napus. Plant CAMTA genes play a crucial role in regulating various processes of plant growth and development, such as those related to flowers, leaves, and roots. Additionally, they are involved in responding to environmental stresses such as drought, salt, cold, and injury, as well as regulating responses to hormone signals like ethylene, abscisic acid (ABA), auxin, salicylic acid (SA), and jasmonic acid (JA) [19,20]. Under low-temperature conditions, Arabidopsis CAMTAs respond to rapid temperature decrease by activating downstream DREB1 expression [21]. Arabidopsis AtCAMTA3, for instance, interacts with 10 transcription factors, including AtCBF2, to positively regulate the expression of the cold stress gene AtCBF2 [22]. Both AtCAMTA1 and AtCAMTA3 exhibit significantly reduced tolerance to freezing [23,24]. AtCAMTA1 and AtCAMTA2 synergistically interact with AtCAMTA3 at low temperature (4 °C), inducing high transcriptional levels of AtCBF1, AtCBF2, and AtCBF3 within 2 h. This interaction promotes the biosynthesis of salicylic acid, enhancing plant cold resistance [23]. Two homozygous T-DNA insertion mutants (camta3-1, camta3-2) of AtCAMTA3 in Arabidopsis show increased spontaneous damage [25]. Overexpression of GmCAMTA12 in Arabidopsis and soybean reveals that they function in the regulation of soybean root development and drought stress [26]. ZmCAMTA4a, ZmCAMTA7a, and ZmCAMTA7b are significantly upregulated in the buds after cold stress treatment in maize, with only ZmCAMTA4a induced by cold stress in the roots [27]. Doherty et al. found that tomato SlCAMTA3 can bind to the conserved motif CCGCGT of SlCBF2, thereby positively regulating SlCBF2. In addition, the other two low-temperature-induced genes, SlCBF1 and ZAT12 (encoding a zinc finger protein), may also be directly regulated by SlCAMTA3 [28]. However, their induction is inhibited by low temperature in the CAMTA1/sr2-CAMTA3/sr1 double mutant, suggesting joint regulation by CAMTA3/SR1 and CAMTA1/SR2 in plant responses to low-temperature stress [29]. Kim et al. observed that among the three functionally redundant CAMTA genes, only CAMTA3 expression was induced by low temperature. This may explain why the CAMTA3 mutant is sensitive to temperature drops when a single gene is missing [23,30]. Researchers also found that CAMT1-3 synergistically promotes the rapid response of CBF1-3 to low temperatures [23,31]. Despite the presence of many stress-related elements in the CAMTA gene promoter region, the upstream regulatory factors governing CAMTA gene expression remain unclear. Additionally, downstream genes regulated by CAMTA are currently limited in number. It is essential to identify target genes regulated by CAMTA on a genome-wide scale, providing a foundation for a comprehensive exploration of CAMTA functions and the analysis of its regulatory role in plant stress response, growth, and development processes.
Eggplant, particularly the purple variety, boasts a higher vitamin content compared to other members of the Solanaceae family, making it a popular and economically significant vegetable crop. However, eggplant is highly susceptible to low temperatures during the planting process, and enhancing its cold resistance has become a key research focus. In recent years, technological advancements, including modern molecular biology methods, high-throughput sequencing, and genetic engineering technologies, have led to continuous improvements in eggplant genome data. This progress has facilitated the extensive screening of low-temperature-tolerant germplasm and the identification of related gene expressions, garnering widespread attention [32,33]. These advancements offer a more in-depth understanding of the cold response mechanism and molecular pathways in eggplant. Against this backdrop, it is crucial to explore additional genes that respond to low temperatures in the eggplant genome. A comprehensive understanding and analysis of the regulatory mechanism of low-temperature stress in eggplant can be achieved through the use of modern technologies. This study conducts a genome-wide identification of CAMTA proteins from the Solanaceae family and examines the structure of CAMTA members at the whole-genome level. The evolutionary relationships among different members of this gene family are investigated, along with their expression patterns in eggplant under cold conditions. In summary, the results of this study aim to provide valuable information for further investigations into the functional and regulatory mechanisms of CAMTA, serving as core components in the Solanaceae family.

2. Results

2.1. Identification and Evolution Analysis of CAMTA Family Members

In this study, a total of 28 CAMTA members were identified from the Solanaceae Genomics database (https://solgenomics.net/, accessed on 14 October 2023) [34]. The number of CAMTA genes ranged from three to seven in different species. The basic physical and chemical properties of the CAMTA family members were analyzed (Table 1). The results showed that the ORF lengths of CAMTAs varied from 2.355 (CaCAMTA4) kb to 3.309 (SmCAMTA2) kb; amino acid lengths ranged from 785 (CaCAMTA4) to 1103 (SmCAMTA2) aa. The molecular weights (MWs) ranged from 88.879 (CaCAMTA4) to 123.588 (SmCAMTA2) kDa, and the theoretical isoelectric point (pIs) ranged from 5.43 (SmCAMTA2) to 7.15 (SmCAMTA1). SmCAMTA1 was predicted to be a basic protein with a theoretical isoelectric point greater than 7 (Table 1). All members were predicted to be located in the nucleus, and none were predicted to contain signal peptides or TMHs. Chromosomal localizations showed that CAMTAs from Solanum melongena, Solanum lycopersicum, Solanum pennellii, Capsicum annuum, and Lycium barbarum were unevenly distributed across eight chromosomes, respectively (Table 1). Some chromosomes contained two CAMTA genes, such as Chr1 and Chr5 in S. melongena; Chr1 and Chr12 in S. lycopersicum; and Chr11 in C. annuum. Nevertheless, Chr1 contained three CAMTA genes in S. pennellii.
To investigate the phylogenetic relationship among Solanaceae CAMTA proteins and those from other plant species, an unrooted phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA 11.0.10 software. The 124 CAMTA genes from 17 species were divided into three subgroups: subgroup I (59 members), subgroup II (36 members), and subgroup III (29 members) (Figure 1). Subgroup I included members from 16 plant species (excluding L. barbarum), subgroup II contained at least one member from all 17 plant species, and subgroup III was the smallest, exclusively containing CAMTAs from 15 plant species. Members of the LbCAMTA family were specifically distributed in subgroup II. In addition, we found that all CAMTA proteins had conserved domains of CG-1, TIG, ANK, and IQ.

2.2. Gene Structure and Conserved Motif Analysis of CAMTA Family Members

Figure 2A illustrates a distinct cluster of Solanaceae CAMTA proteins, aligning with the phylogenetic tree constructed using CAMTA sequences from 17 plant species (Figure 1, Table S1). To infer structural variations and potential functional divergence, the coding sequences of CAMTA family genes were analyzed using the MEME tool (http://meme-suite.org/, accessed on 17 April 2020) [35]. The result showed that the CAMTA protein family contains 12 conserved motifs, named motifs 1–12 (Figure 2A,B). The number of motifs varied from 9 to 12. All CAMTA proteins contained motif 2, motif 3, motif 4, motif 5, motif 6, motif 7, motif 9, and motif 11; however, motif 12 was only present in subgroup II of CAMTA. Motif 7, motif 11, and motif 12 were unknown, and the Pfam 36.0 database (http://pfam.xfam.org/, accessed on 8 January 2021) was unable to find the corresponding domain. Motifs 1, 3, and 10 were associated with the CG-1 domain, motifs 2 and 6 with the ANK domain, and motifs 8 and 5 with the TIG domain, while motifs 4 and 9 were related to the IQ domain. Some motifs were specific to members of particular subgroups, suggesting subgroup-specific functions.
To comprehend the structural characteristics of CAMTA genes, exon–intron structures were analyzed. TBtools v2.042 software (https://github.com/CJ-Chen/TBtools-II/tree/2.042/, accessed on 21 September 2023) was employed for this analysis, and the gene structure map of the CAMTA family was generated. The number of introns varied from 5 to 14, with SmCAMTA2 and SmCAMTA6 possessing 14 introns each. SpCAMTA6 and LcCAMTA2 had fewer introns, namely five and eight, respectively. Notably, introns and exons in the gene structures of subgroup II members within the same branch exhibited significant differences. Although the introns of Solanaceae CAMTA genes differed, members with the highest homology, such as SpCAMTA1, SmCAMTA5, and SlCAMTA3, displayed similar gene structures, intron lengths, and the same number of exons. Exon–intron organization was generally consistent within the same group, supporting their close evolutionary relationships (Figure 3).

2.3. Analysis of Cis-Regulatory Element of CAMTA Family Promoter

To gain insight into the potential functions of CAMTA genes in eggplant, cis-regulatory elements were predicted using the PlantCARE online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 22 July 2022) within the 2000 bp promoter region of each gene [36]. Various cis-regulatory elements were identified in the promoter regions of SmCAMTAs, including elements related to biological and abiotic stress responses (11), plant hormone signaling (8), and growth and development (4), indicating the involvement of CAMTA genes in various stress responses. The highest number of biological and abiotic stress responses mainly include GATA-box (SmCAMTA2), G-box (SmCAMTA1, SmCAMTA 3, and SmCAMTA4), TCT motifs (SmCAMTA6), Box-4 (SmCAMTA2, SmCAMTA3, and SmCAMTA4), and LTR (SmCAMTA1, SmCAMTA2, and SmCAMTA5) (Figure 4). Additionally, several drought-responsive cis-elements (MYC) were present in almost all SmCAMTAs. There were eight phytohormone response elements in the promoter region: TCA responds to salicylic acid (SmCAMTA1 and SmCAMTA2); ABRE responds to abscisic acid (SmCAMTA4 and SmCAMTA5); AuxRR-core respond to auxin (SmCAMTA5); and TGACG-motif and CGTCA-motif respond to methyl jasmonate (SmCAMTA1, SmCAMTA2, SmCAMTA3, SmCAMTA4, and SmCAMTA5). Moreover, growth and development elements such as O2-site, MBS1, and MYB were identified in five genes (SmCAMTA1, SmCAMTA3, SmCAMTA5, and SmCAMTA6). These results underscore the diverse roles of each SmCAMTA in coping with diurnal changes, hormonal responses, and various stresses.

2.4. Expression Profiling of SmCAMTAs Genes in Different Tissues and under Cold Stress

To elucidate the functional roles of SmCAMTA genes in eggplant during various developmental stages, the expression patterns of SmCAMTAs were analyzed across different tissues, including root, leaf, and flower, based on RNA-seq data from a previous study [37] (Figure 5A). SmCAMTA genes displayed diverse tissue-specific expression patterns. SmCAMTA3/4 were lowly expressed in root tissue and highly expressed in fruit tissue. The expression levels of SmCAMTA1 decreased in the root and increased in the leaf and fruit. SmCAMTA2/6 genes were only expressed in leaf tissue. Notably, SmCAMTA5 did not exhibit detectable expression in the tissue-specific transcriptome.
In the early stages of cold stress, the transcriptomes of both cold-sensitive (‘E7134’) and cold-tolerant (‘E7145’) eggplant varieties were analyzed. The spatiotemporal expression patterns of SmCAMTA genes under abiotic cold stress conditions were investigated based on transcriptomic data. As shown in Figure 5B, SmCAMTAs were differentially expressed under cold stress conditions. Specifically, all SmCAMTAs were induced to some extent by cold at different time points, with SmCAMTA1/3/4/5 being slightly affected by cold, while SmCAMTA2 and SmCAMTA6 transcripts significantly increased within 7 d. These genes actively responded to cold stress, and their transcriptional abundance was notably higher in the cold-tolerant type compared to the cold-sensitive type.
We further explored the expression patterns between the cold-resistant variety ‘E7134’ and the cold-sensitive variety ‘E7145’ under cold stress conditions using qRT-PCR. As shown in Figure 5C, the expression patterns of SmCAMTAs varied at different times and in different species. Except for SmCAMTA2 of ‘E7145’, other genes were upregulated under cold treatment an 0− 1 d, and then the expression levels of SmCAMTAs were divided into three categories from 1 d to 7 d under cold treatment time: the transcription levels of SmCAMTA1/3 first increased and then significantly decreased at 2 d; the transcription levels of SmCAMTA4/5 significantly decreased persistently after 2 d; and the transcription levels of SmCAMTA2/6 significantly increased persistently throughout the cold stress treatment. In addition, in the early stage of cold stress, the expression level of SmCAMTA1/2 in ‘E7145’ was higher than that in ‘E7134’, and at 4–7 days, the expression levels of SmCAMTA1/4 in ‘E7145’ were higher than that in ‘E7134’. During the entire cold stress period, the expression level of SmCAMTA3/5/6 in ‘E7134’ was higher than that in ‘E7145’. Importantly, the expression levels of SmCAMTA2 and SmCAMTA6 were more than 4.1-fold and 3.9-fold higher after 7 days under cold stress, respectively. This result shows that the similar expression patterns of SmCAMTA2/6 may have a positive contribution to the cold resistance of eggplants.

2.5. Protein–Protein Interaction Analysis of SmCAMTAs

Biological macromolecules typically engage in physiological functions through interactions with other proteins, forming complexes. Therefore, investigating these interaction relationships is crucial for understanding the function of CAMTA. The whole-transcriptome protein interaction network of eggplant was constructed using homologous mapping and domain interaction methods with Arabidopsis as the reference species. The subnetwork of the SmCAMTA2 core protein was extracted (Figure 6A and Table S4). A total of 62 non-redundant potential interacting proteins of SmCAMTA2 were identified, comprising nine types of transcription factors (TFs) (C2H2, GRAS, bHLH, MYB, WRKY, TCP, AP2/ERF, NAC, DOF), three kinases (phosphoribulo, lectin, and shikimate kinases), as well as several enzymes and RNA-binding proteins. In addition, 93.5% of interacting protein genes were positively correlated with SmCAMTA2 (p < 0.05) under cold stress, and GO enrichment analysis showed that they were related to “DNA binding”, “hormone metabolism processes”, “photosynthesis”, and “catalytic activity” functions (Figure 6B). Therefore, it is suggested that SmCAMTA2 may recruit plant hormone-related proteins under cold stress. Notably, a strong interaction was observed between SmERF1, SmCAMT6, and SmCAMTA2 (p < 0.001) (Figure 6A).
A GAL4-based yeast two-hybrid (Y2H) system and bimolecular fluorescence complementation (BiFC) were employed to investigate the relationships between SmCAMTA2, SmCAMTA6, and SmERF1. As shown in Figure 7A, the positive control and experimental group transformants grew on SD/- Trp- His-Ura medium containing X-a-gal and turned blue, indicating that SmCAMTA2 interacts with SmCAMTA6 and SmERF1. Subsequently, a BiFC experiment was conducted on tobacco leaves (Figure 7B), confirming the interaction between SmCAMTA2 and SmERF1, as well as SmCAMTA6. However, there was no interaction observed between SmCAMTA6 and SmERF1.

2.6. SmCAMTA2 and SmCAMTA6 Regulated the Transcription of Cold-Related CBF Genes under Cold Stress

To investigate the functions of SmCAMTA2 and SmCAMTA6 and their potential transcriptional activation activity, trans-activation assays were conducted on transcription factors. In the yeast system, both SmCAMTA2-BD and SmCAMTA6-BD yeast cells, as well as positive controls, exhibited robust growth on selective media (SD/- Trp/- His + X-α-gal) and turned blue. Conversely, negative controls (BD) could not grow on selective media (Figure 8A). This observation provides additional evidence supporting the role of SmCAMTA2 and SmCAMTA6 in activating transcription factors.
The AtCBFs cold response pathway plays a central role in cold acclimation [38]. Previously, it was reported that the expressions of DREB1/CBF genes were regulated by many transcription factors, indicating that the expression regulation of these genes was highly complex. To explore the transcriptional regulation of SmCBFs by SmCAMTA2 and SmCAMTA6, and the involvement of SmERF1 in the regulation of SmCBFs genes, yeast one-hybrid experiments and the LUC/REN reporting system were employed. The results indicated that the co-transformed single colonies of the SmCBF control group grew normally on SD/- Leu/- Trp medium without 3-AT inhibitors. In contrast, the yeast colonies of the negative control group did not grow on a plate containing 25 mM 3-AT, while the colonies of the positive experimental group exhibited normal growth in size and morphology (Figure 8B). In addition, the co-transformation of SmCBFs pro LUC and pGreenII 62-SK into tobacco leaves showed very low luciferase luminescence, while luminescence was significantly enhanced through the co-transformation of SmCBF2 pro LUC with SmCAMTA2-62-SK and SmCAMTA2-62-SK. LAR confirmed that SmCAMTA2 and SmCAMTA6 activated the SmCBF2 promoter in tobacco leaves (Figure 8C). Furthermore, the LUC/REN ratio showed a significant, approximately twofold increase when SmERF1-62-SK and SmCAMTA2-62-SK were co-presented, with no significant change observed when they were co-present with SmCAMTA6-62-SK. These results suggest that SmERF1 promotes the expression of CBF2 by interacting with SmCAMTA2 and binding to the promoter element of SmCBF2.

3. Discussion

CAMTA transcription factors play a pivotal role in calcium/calmodulin signaling pathways, mediating gene transcriptional regulation, which is an essential process for plants to respond to exogenous hormones and abiotic stress [39,40,41]. Eggplant development exhibits distinct specificity, with growth stimulated by various biotic and abiotic stressors and hormones. This suggests a potential crucial biological function for CAMTA in eggplant. With the completion of genome sequencing for eggplant, we were able to conduct a comprehensive analysis of CAMTA. In this study, we identified the CAMTA gene family of Solanaceae at the whole-genome level. The number of CAMTAs ranged from three to seven. The copy number of the homologous CAMTA gene family generally varies among different species, which is caused by different gene gain and loss rates. The copy number variation of gene families provides a genetic basis for the innovation and diversification of species phenotypes and is closely related to the evolution of genome size and species differentiation in organisms. In this study, we comprehensively analyzed the gene structure and phylogeny of the CAMTA gene family. The promoter elements and transcriptional expression patterns of eggplant CAMTAs were also analyzed. Twenty-eight CAMTA genes were identified in Solanaceae. HMMER 3.0 (hidden Markov model, HMM) online software (http://eddylab.org/software/hmmer3/3.0/, accessed on 15 May 2018) software and the blastp program of the hidden Markov model were employed to identify CAMTA members of other species at the whole-genome level. The intersection of the results of the two outputs was taken for further conservative domain analysis. Finally, CAMTA members confirmed to contain the CG-1 domain were identified.
The phylogenetic tree results show that the closer the clustering relationship, the more likely it is to have similar functions [42]. Phylogenetic analysis revealed that CAMTA genes can be divided into three subgroups, and the distribution of Arabidopsis CAMTAs in other phylogenetic groups aligns with that of this specific phylogenetic group. The motif composition of CAMTA proteins in Solanaceae is highly conservative, indicating a relatively consistent function for CAMTAs. Notably, some genes lack certain motifs, potentially contributing to the functional diversity observed in CAMTA genes. Gene structure analysis identified a high similarity in the CAMTA homeotic genes of Solanaceae, suggesting a highly conservative gene structure [43,44,45]. The relative position and number of introns within the same branch were also conservative, but variations across different branches contributed to the diversification of gene function. Genetic phylogenesis, gene classification, and gene structure analysis can provide valuable insights for more accurate and convenient exploration of similar gene families and their functions.
The function of CAMTA genes in Arabidopsis has been extensively studied, providing insights into the potential functions of related genes in Solanaceae. Evolutionarily related genes often share similar functions. For instance, AtCAMTA3 in Arabidopsis positively regulates the CBF2 gene, playing a crucial role in plant low-temperature stress response, and influencing the biosynthesis of salicylic acid (SA) [23]. SmCAMTA2 and SmCAMTA6 were significantly and continuously upregulated by low-temperature stress; it was speculated that SmCAMTA2 and SmCAMTA6 may play a role in low-temperature stress through biochemical reactions similar to AtCAMTA3. At the same time, AtCAMTA3 was also a drought stress response factor [46], suggesting that SmCAMTA2 and SmCAMTA6 may also have functional diversity. Pandey et al. found through gene chip research that 17 genes related to auxin were upregulated in AtCAMTA1 mutants [39]. Both AtCAMTA1 mutants and RNAi-mediated CAMTA1 inhibitory transgenic lines displayed a phenotype of auxin hypersensitivity to hypocotyl elongation, indicating the involvement of AtCAMTA1 in auxin signaling pathways and its role in regulating plant growth and development. Yang et al. observed differential expression of seven CAMTA genes during tomato fruit development and maturation, suggesting their potential role in the regulation of tomato fruit development [47]. It was speculated that CAMTA homeotic genes in eggplant were involved in the growth and development of tissues and organs. Research in Arabidopsis showed that AtCAMTA2 is an activated transcription factor of AtALMT1 (aluminum activated malic acid transporter 1) [48], and AtCAMTA3 plays a negative regulation role in SA-mediated plant defense response [30,49]. The loss of repression of AtCAMTA1, AtCAMTA2, and AtCAMTA3 can induce the initiation of plant defense genes and systemic resistance [23]. MeCAMTA3 in cassava regulated the resistance of cassava to bacterial wilt disease by regulating various immune responses during the interaction between cassava and Xanthomonas flavescens [50]. Therefore, it is speculated that SmCAMTA4 and SmCAMTA5 genes exhibit functional diversity and may be involved in hormone signal transduction, growth and development regulation, low-temperature stress response, salt stress, and defense response. Among them, SmCAMTA2 and SmCAMTA6 genes are identified as important candidate genes for further studying the molecular mechanism of eggplant response to low-temperature stress. This study represents the first systematic analysis of the CAMTA gene, laying the foundation for further research on the function of the CAMTA gene in Solanaceae.
The results of protein interaction network analysis revealed a complex regulatory network involving directly functional proteins which regulated plant growth and development through transcriptional regulation of SmCAMTAs and other functional genes. SmCAMTA2 and SmCMATA6 exhibited high expression levels under cold conditions (Figure 5B,C), suggesting a potential role in binding to the CM cis-element in the SmCBF2 promoter. This binding could regulate the expression of downstream CBF genes, similar to their orthologs such as AtCAMTA3 and AtCAMTA2. Similarly, the interaction analyses of SmCAMTAs demonstrated that SmCAMTA2 and SmCAMTA6 were directly related to CBF/DREB1C. It is important that the interaction of SmCAMTA2 and SmERF1 promoted the transcriptional activity of SmCBF2, which may be an important mechanism in eggplant’s response to cold stress. Previous studies have shown that AtCAMTA1 and AtCAMTA2 work synergistically with CAMTA3 at low temperatures (4 °C), inducing high transcriptional expression of CBF1, CBF2, and CBF3, thereby enhancing plant frost resistance. Additionally, AtCAMTA1, AtCAMTA2, and AtCAMTA3 collectively inhibit SA biosynthesis at warm temperatures (22 °C) [23]. Functional analyses using AtCAMTA mutants indicated that AtCAMTA3 negatively regulated immunity triggered by flg22 [30]. SmCAMTA1 and SmCAMTA5 were an ortholog of AtCAMTA5, which speculates that its function may be related to the pollen pollination and development of eggplant [51]. It has been reported that the CAMTA family is involved in fruit development and abiotic stress processes in tomato [46], soybean [26], and other plants [52]. In tomato, for example, SlCAMTA3 expression is scarce in leaves in the seedling and flowering stages, while it is highly expressed in roots in the seedling stage [53]. In the case of eggplant, the expression of the SmCAMTA3 and SmCAMTA4 genes is highly observed in the leaves but is lowly detected in eggplant flowers (Figure 5A). The results of protein–protein interaction network analysis indicate that these three proteins are directly related to each other, suggesting their potential collaborative role in responding to cold stress during the reproductive growth stage of eggplant.

4. Methods and Materials

4.1. Plant Materials, Growth Conditions, and Stress Treatments

Two varieties of S. melongena, ‘E7134’ (a cold-tolerant variety) and ‘E7145’ (a cold-sensitive variety), were utilized for cold treatment in this study. Both eggplant materials were preserved and provided by the Sichuan Academy of Agricultural Sciences. Seedlings were cultivated in a greenhouse under controlled conditions at 26 ± 1 °C, 65% relative humidity, and a 16/8 h light/dark period in the agricultural science area of Sichuan Province (Chengdu, China). For the cold treatment, seedlings with 5 to 6 leaves were transplanted into plastic pots. Subsequently, all seedlings were exposed to cold stress for 7 days, maintaining a temperature of 5 °C during the day and 10 °C during the night. Sampling was conducted at 0, 1, 2, 4, and 7 days after the initiation of cold stress, with three biological replicates collected at each time point. The samples from both S. melongena varieties’ seedlings were promptly frozen in liquid nitrogen and stored at −80 °C for subsequent analyses.

4.2. Identification of CAMTA Genes

All CAMTA genes in the complete Solanaceae genome were identified from Solanaceae species (https://solgenomics.net/, accessed on 14 October 2023) using HMMER 3.0 online software (http://eddylab.org/software/hmmer3/3.0/, accessed on 15 May 2018) [54]. HMM profiles for the CAMTA gene family (Pfam03859: CG-1; Pfam12796: ankyrin repeats; Pfam01833: TIG domain; and Pfam00612: IQ) were obtained from Pfam 36.0 (http://pfam.xfam.org/, accessed on 8 January 2021) [55]. The whole-genome sequences of the other 16 plants (C. annuum, A. thaliana, S. lycopersicum, O. sativa, V. vinifera, S. bicolor, C. lanatus, T. cacao, Z. mays, I. trifida, P. trichocarpa, G. hirsutum, S. pennellii, L. barbarum, B. rapa, and G. max) were downloaded from the Plant JGI Database phytozome v13 (https://phytozome.jgi.doe.gov/pz/portal.html, accessed on 6 October 2022) [56]. These profiles served as queries against whole-genome peptide sequences of A. thaliana retrieved from whole-genome sequence data. After eliminating redundant sequences, the identity of CAMTA proteins containing functional protein domains was confirmed using NCBI-CDD (National Center for Biotechnology Information—Conserved Domain Database, https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 10 September 2021) [57]. Any sequences with missing structural domains were excluded. To understand the genomic organization of CAMTA genes, information on their chromosomal positions was extracted from gff3 files and visualized using TBtools v2.042 (https://github.com/CJ-Chen/TBtools-II/tree/2.042/, accessed on 21 September 2023) [58]. A detailed list of accession names for CAMTA genes is provided in Table 1.

4.3. Phylogenetic Analysis and Classification

The amino acid sequences of the 124 identified CAMTA proteins (refer to Table S3) were utilized for phylogenetic analysis. Multiple sequence alignments of all selected CAMTA sequences were conducted using the MUCLE method with default parameters [59]. Subsequently, unrooted phylogenetic trees of the CAMTA proteins were constructed using MEGA 11.0.10 software (https://www.megasoftware.net/, accessed on 23 April 2021) [60]. The parameters included Poisson correction, pairwise deletion, and a bootstrap test with 1000 replicates. Furthermore, the phylogenetic tree was visually enhanced using EvolView v3 (https://www.evolgenius.info//evolview/#login, accessed on 26 May 2020) [61].

4.4. Motif Composition and Gene Structure Analysis

To identify additional conserved motifs beyond the CAMTA proteins, the Multiple Em for Motif Elicitation tool (http://meme-suite.org/tools/meme, accessed on 17 December 2022) [35] was employed. Specific parameters, including maximum width, minimum width, and maximum number of motifs, were set at 5, 150, and 12, respectively. The motifs were sequentially numbered based on their order in MEME. Signatures common to genes within one of the three similarity groups were assigned as group-specific motifs. The outcomes were visualized using TBtools v2.042 (https://github.com/CJ-Chen/TBtools-II/tree/2.042/, accessed on 21 September 2023) [58] to protein structures. The CDS and DNA sequences of CAMTAs were obtained from Solanaceae species (https://solgenomics.net/, accessed on 14 October 2023) and GSDS v2.0 (Gene Structure Display Server, http://gsds.cbi.pku.edu.cn/, accessed on 27 May 2020) [59] was utilized to analyze gene structures, providing a comprehensive visualization of the exon–intron organization of CAMTA genes.

4.5. Analysis of Cis-Acting Elements in the Promoter Region

The PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 22 July 2022) was utilized to identify cis-acting elements within the 2000 bp sequence upstream of the transcription start site of SmCAMTA genes [36]. The distribution map of cis-acting elements was visualized via TBtools v2.042 (https://github.com/CJ-Chen/TBtools-II/tree/2.042/, accessed on 21 September 2023) [58].

4.6. Gene Expression Analysis

Expression data for SmCAMTA family members from different tissues were obtained from the China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA013268) and are publicly accessible at https://ngdc.cncb.ac.cn/gsa. Log2 (1 + FPKM (treatment)/1 + FPKM (control)) were calculated using FPKM to indicate the fold change in gene expression levels. Subsequently, the expression levels of selected plant SmCAMTA genes were analyzed using TBtools v2.042 (https://github.com/CJ-Chen/TBtools-II/tree/2.042/, accessed on 21 September 2023) [58].

4.7. RNA Isolation and qRT-PCR Analysis

Total RNA was extracted from leaves subjected to different abiotic stresses using the RNA Prep Pure Plant Kit (Tiangen Biochemical Technology, Beijing, China: DP201101X). The concentration and purity of the RNA were assessed using a Nanodrop micro spectrophotometer (Thermo Scientific, Waltham, MA, USA) and agarose gel electrophoresis. The first strand of cDNA, derived from mRNA, was synthesized using HiScript®Q RT SuperMix (Vazyme, Nanjing, China). Quantitative real-time PCR (qRT-PCR) was performed using SYBR-green fluorescence with a QuantStudioTM Real-Time PCR System (Thermo Fisher Scientific). Six CAMTA genes were selected to validate expression patterns under cold stress. The data were normalized by the internal control gene β-actin and the 2−∆∆CT analysis method was utilized to calculate relative expression levels [62]. The data were finally visualized as mean values ± standard error (±SE). Primers are listed in the Table S2.

4.8. Protein–Protein Analysis of SmCAMTAs

For the yeast two-hybrid experiment, the coding regions of SmCAMTA2 and SmERF1 were amplified and fused in-frame downstream of the GAL4 DNA-binding domain in the pGBKT7 vector, while SmCAMTA6 was fused in the pGADT7 vector. Primers are listed in Table S2. The constructs were transformed into strain AH109 (Saccharomyces cerevisiae) and grown on SD/- Trp and SD/- Trp/- His/- Ura medium, respectively. Autoactivation activity was examined on SD/- Trp/- His/- Ura medium containing X-a-gal. The pGBKT7-53+pGADT7-T vector and pGBKT7-Lam+pGADT7 were used as positive and negative controls, respectively.
For the bimolecular fluorescent complementation of the SmCAMTA2/6 transcription factors in intact cells, the plant transient expression vectors pXY103 (cYFP) and pXY104 (nYFP) were utilized. A transient transformation system was established using 1M MgCl2 and 0.5 M acetosyringone. The construct recombinant plasmids SmCAMTA2-cYFP and SmCAMTA6-nYFP were then transferred into tobacco leaves using Agrobacterium strain (GV3101), with SmCAMTA2-cYFP+nYFP and cYFP+SmCAMTA6-nYFP as the control. The fluorescence of fusion proteins was detected by laser scanning confocal microscopy, Leica (Zeiss, Jena, Germany).

4.9. SmCAMTAs Participate in Transcriptional Regulation of SmCBFs

The effector vector pGADT7-SmCAMTAs and the reporter vector pHIS2-SmCBFs pro were constructed and co-transformed into the yeast strain Y187. The transformed yeast cells were cultured at 28 ℃ on SD (SD/-Trp/-Leu) medium until colonies grew normally. After dilution based on a gradient, they were applied to SD (SD/-Trp/-Leu/-His) medium with a final concentration of 20 mM 3-AT inhibitor for inverted cultivation, and colony morphology was observed. The primers used are listed in Table S3.
The promoter sequences of SmCBF genes were cloned into the pGreenII 0800-LUC double-reporter vector. The full coding sequences (CDS) of SmCAMTA2, SmCAMTA6, and SmERF1 were then inserted into the pGreenII 62-SK effector vector. Co-infiltration of the reporter and effector plasmids was performed in Nicotiana benthamiana leaves [63]. The Dual-Luciferase Assay Kit (Promega, Madison, WI, USA) was utilized to analyze luciferase activity. Results are expressed as the LUC (firefly luciferase)/REN (renilla luciferase) ratio, representing an average of six replicates.

5. Conclusions

This study identified six members of the CAMTA gene family in Solanaceae through genome-wide analysis, categorizing them into three subgroups—subgroup I, subgroup II, and subgroup III—based on their phylogenetic relationships. Comprehensive analyses of phylogenetics, gene structure, and motif composition were conducted in Solanaceae. Expression pattern analyses of SmCAMTAs revealed distinct expression profiles in various tissues of eggplant. RNA-seq data and qRT-PCR results indicated that SmCAMTA2/6 genes positively responded to cold stress, and the interaction between SmCAMTA2 and SmERF1 facilitated the transcription of SmCBF2. These findings offer crucial insights into the significant role of CAMTA in responding to cold stress and lay the groundwork for further exploration of the functional aspects of Solanaceae CAMTA gene family members.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25042064/s1.

Author Contributions

C.F. and P.C. conceived and designed this study. P.C. and Y.L. (Yanhong Lan) were involved in data interpretation. P.C., F.G., C.L., Y.L. (Yifan Li) and F.X. organized and performed the experiments. Y.L. (Yanhong Lan) and P.C. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from ‘Breakthrough vegetable breeding materials, innovation methods and new variety breeding’, Sichuan Provincial Vegetable Breeding Key project (grant No. 2021YFYZ0022); the ‘Breeding Research and Development’ project of the Molecular Breeding Platform for Major Economic Crops (grant No. 2021YFYZ0010); and the 1 + 9 Program of SAAS (1+9KJGG03).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The transcriptome raw data are available from the online web site China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (https://ngdc.cncb.ac.cn/gsa accessed on 6 Jan 2023, GSA: CRA0132687).

Acknowledgments

The authors are grateful to Wang Yikui’s team from Guangxi Academy of Agricultural Sciences (Guangxi, China) for providing eggplant genome information.

Conflicts of Interest

The authors declare no conflict of interests.

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Figure 1. Phylogenetic analysis of SmCAMTAs and known CAMTAs of 18 plant species. Species abbreviations are as follows: Capsicum annuum (Ca), Arabidopsis thaliana (At), Solanum lycopersicum (Sl), Oryza sativa (Os), Vitis vinifera (Vv), Solanum melongena (Sm), Sorghum bicolor (Sb), Citrullus lanatus (Cl), Theobroma cacao (Tc), Zea mays (Zm), Ipomoea trifida (It), Populus_trichocarpa (Pt), Gossypium hirsutum (Gh), Solanum pennellii (Sp), Lycium barbarum (Lb), Brassica rapa (Br), and Glycine_max (Gm).
Figure 1. Phylogenetic analysis of SmCAMTAs and known CAMTAs of 18 plant species. Species abbreviations are as follows: Capsicum annuum (Ca), Arabidopsis thaliana (At), Solanum lycopersicum (Sl), Oryza sativa (Os), Vitis vinifera (Vv), Solanum melongena (Sm), Sorghum bicolor (Sb), Citrullus lanatus (Cl), Theobroma cacao (Tc), Zea mays (Zm), Ipomoea trifida (It), Populus_trichocarpa (Pt), Gossypium hirsutum (Gh), Solanum pennellii (Sp), Lycium barbarum (Lb), Brassica rapa (Br), and Glycine_max (Gm).
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Figure 2. Comparative analysis of conserved motif of CAMTAs family. (A) Phylogenetic relationships and conserved protein motifs of CAMTA family. (B) Protein sequence of conserved motif.
Figure 2. Comparative analysis of conserved motif of CAMTAs family. (A) Phylogenetic relationships and conserved protein motifs of CAMTA family. (B) Protein sequence of conserved motif.
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Figure 3. Structural analysis of CAMTA gene family members in Solanaceae. Blue color bars are the UTR regions, yellow color bars are the CDS regions, and solid black lines represent introns.
Figure 3. Structural analysis of CAMTA gene family members in Solanaceae. Blue color bars are the UTR regions, yellow color bars are the CDS regions, and solid black lines represent introns.
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Figure 4. Distribution and function prediction of cis-acting elements in the upstream 2 kb regions of SmCAMTA genes. The different colors and numbers of the grids indicate the number of different cis-acting regulatory elements in these SmCAMTA genes.
Figure 4. Distribution and function prediction of cis-acting elements in the upstream 2 kb regions of SmCAMTA genes. The different colors and numbers of the grids indicate the number of different cis-acting regulatory elements in these SmCAMTA genes.
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Figure 5. Expression patterns of SmCAMTAs under cold stress conditions. Samples at 0 d were set as controls, and the data were calculated by using 2−ΔΔCt method and FPKM. Data are shown as means ± SE (n = 3). (A) Expression profiles of SmCAMTAs in various developmental stages. (B) Relative expression levels of SmCAMTAs under cold stress. (C) Relative expression levels of SmCAMTAs under cold treatment at five time points by qRT-PCR.
Figure 5. Expression patterns of SmCAMTAs under cold stress conditions. Samples at 0 d were set as controls, and the data were calculated by using 2−ΔΔCt method and FPKM. Data are shown as means ± SE (n = 3). (A) Expression profiles of SmCAMTAs in various developmental stages. (B) Relative expression levels of SmCAMTAs under cold stress. (C) Relative expression levels of SmCAMTAs under cold treatment at five time points by qRT-PCR.
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Figure 6. The protein–protein interaction network of CAMTA genes. Network nodes represent proteins; filled nodes represent proteins with known or predicted 3D structures. Edges represent protein–protein associations. Different colors represent various types of interactions.
Figure 6. The protein–protein interaction network of CAMTA genes. Network nodes represent proteins; filled nodes represent proteins with known or predicted 3D structures. Edges represent protein–protein associations. Different colors represent various types of interactions.
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Figure 7. Interaction relationship detected between SmCAMTA2 and SmCAMTA6 using GAL4-based Y2H system and bimolecular fluorescence complementation. (A) Yeast two-hybrid experiment on SmCAMTAs. The interaction relationship was examined on SD/- Trp/- Leu/- His/− Ade medium containing X-a-gal. The pGBKT7-53+pGADT7-T vector and pGBKT7-Lam+pGADT7 were used as positive and negative controls, respectively. (B) In BiFC assays, SmCAMTA2-nYFP, cYFP, and nYFP and SmCAMTA6-cYFP, SmCAMTA2-nYFP, and SmCAMTA6-cYFP were co-transformed in N. benthamiana leaf cells, respectively; bar = 75 µm. Bright field, GFP field, and merged field indicate the state of fluorescent protein under three different channels.
Figure 7. Interaction relationship detected between SmCAMTA2 and SmCAMTA6 using GAL4-based Y2H system and bimolecular fluorescence complementation. (A) Yeast two-hybrid experiment on SmCAMTAs. The interaction relationship was examined on SD/- Trp/- Leu/- His/− Ade medium containing X-a-gal. The pGBKT7-53+pGADT7-T vector and pGBKT7-Lam+pGADT7 were used as positive and negative controls, respectively. (B) In BiFC assays, SmCAMTA2-nYFP, cYFP, and nYFP and SmCAMTA6-cYFP, SmCAMTA2-nYFP, and SmCAMTA6-cYFP were co-transformed in N. benthamiana leaf cells, respectively; bar = 75 µm. Bright field, GFP field, and merged field indicate the state of fluorescent protein under three different channels.
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Figure 8. Identification of the interaction between SmCAMTA2/6 and SmCBFs promoters. (A) Transcriptional activation associated with SmCAMTA2/6 in yeast cells. (B) Interaction between SmCAMTA2/6 and SmCBFs promoters system. pHIS2+pGADT7-Rec2-53 and p53HIS2+pGADT7-Rec2-53 were the negative control and positive control, respectively. (C) Calculation of structure and LUC/REN ratio used in transient trans activation analysis as the final transcriptional activity. Blank carriers are used as effectors in control experiments. Using the promoter of CBF in LRA, each value represents the average of three biological replicates. Values represented as mean ± standard deviation, and p values of <0.01 (**), <0.001 (***), and <0.001 (****) were considered to be significant statistically.
Figure 8. Identification of the interaction between SmCAMTA2/6 and SmCBFs promoters. (A) Transcriptional activation associated with SmCAMTA2/6 in yeast cells. (B) Interaction between SmCAMTA2/6 and SmCBFs promoters system. pHIS2+pGADT7-Rec2-53 and p53HIS2+pGADT7-Rec2-53 were the negative control and positive control, respectively. (C) Calculation of structure and LUC/REN ratio used in transient trans activation analysis as the final transcriptional activity. Blank carriers are used as effectors in control experiments. Using the promoter of CBF in LRA, each value represents the average of three biological replicates. Values represented as mean ± standard deviation, and p values of <0.01 (**), <0.001 (***), and <0.001 (****) were considered to be significant statistically.
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Table 1. Basic physical and chemical analysis of SmCAMTAs. ORF, Open Reading Fame; AA, number of amino acid residues; MW, Molecular Weight; pI, theoretical isoelectric point; Chr, chromosome location.
Table 1. Basic physical and chemical analysis of SmCAMTAs. ORF, Open Reading Fame; AA, number of amino acid residues; MW, Molecular Weight; pI, theoretical isoelectric point; Chr, chromosome location.
SpeciesGene IDNameSubgroupORF (bp)AApIMW (kDa)Chr
Solanum melongenaSMEL4.1_01g027530.1.01SmCAMTA1Subgroup I27699237.15104.2001
SMEL4.1_11g009470.1.01SmCAMTA2Subgroup III330911035.43123.58811
SMEL4.1_03g010310.1.01SmCAMTA3Subgroup II28899635.83107.8493
SMEL4.1_05g010190.1.01SmCAMTA4Subgroup I28659556.48106.5755
SMEL4.1_05g021930.1.01SmCAMTA5Subgroup I27039016.78102.2945
SMEL4.1_01g009270.1.01SmCAMTA6Subgroup III314710495.65118.5631
Solanum lycopersicumSolyc01g057270.2.1SlCAMTA1Subgroup I28719578.92107.7851
Solyc01g105230.2.1SlCAMTA2Subgroup I312010405.47117.4171
Solyc04g056270.2.1SlCAMTA3Subgroup III306310215.76114.1464
Solyc05g015650.2.1SlCAMTA4Subgroup II28059355.89105.3045
Solyc12g035520.1.1SlCAMTA5Subgroup II29199736.25108.98212
Solyc12g099340.1.1SlCAMTA6Subgroup III29459156.74103.93912
Solanum pennelliiSopen01g021880.1SpCAMTA1Subgroup I28359459.02106.5061
Sopen01g026290.1SpCAMTA2Subgroup III27609208.07104.0791
Sopen01g047740.1SpCAMTA3Subgroup I314710495.45118.4781
Sopen04g025150.1SpCAMTA4Subgroup I329410985.51122.8264
Sopen05g011110.1SpCAMTA5Subgroup II28929645.72108.2635
Sopen12g015500.1SpCAMTA6Subgroup II29199736.25108.92212
Sopen12g034010.1SpCAMTA7Subgroup III27309106.66103.2302
Capsicum annuumCA08g15740CaCAMTA1Subgroup I305410185.81114.7988
CA11g07570CaCAMTA2Subgroup II306910236.19115.20011
CA11g10930CaCAMTA3Subgroup III23557856.0188.87911
Lycium barbarumXP_060191074.1LbCAMTA1Subgroup II28389466.24105.81111
XP_060191073.1LbCAMTA2Subgroup II28269626.04107.50811
XP_060191072.1LbCAMTA3Subgroup II28989666.13107.78311
XP_060191071.1LbCAMTA4Subgroup II300310016.09111.97711
XP_060191070.1LbCAMTA5Subgroup II302410086.04112.83611
XP_060191069.1LbCAMTA6Subgroup II303310116.09113.15011
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Cai, P.; Lan, Y.; Gong, F.; Li, C.; Xia, F.; Li, Y.; Fang, C. Identification and Molecular Characterization of the CAMTA Gene Family in Solanaceae with a Focus on the Expression Analysis of Eggplant Genes under Cold Stress. Int. J. Mol. Sci. 2024, 25, 2064. https://doi.org/10.3390/ijms25042064

AMA Style

Cai P, Lan Y, Gong F, Li C, Xia F, Li Y, Fang C. Identification and Molecular Characterization of the CAMTA Gene Family in Solanaceae with a Focus on the Expression Analysis of Eggplant Genes under Cold Stress. International Journal of Molecular Sciences. 2024; 25(4):2064. https://doi.org/10.3390/ijms25042064

Chicago/Turabian Style

Cai, Peng, Yanhong Lan, Fangyi Gong, Chun Li, Feng Xia, Yifan Li, and Chao Fang. 2024. "Identification and Molecular Characterization of the CAMTA Gene Family in Solanaceae with a Focus on the Expression Analysis of Eggplant Genes under Cold Stress" International Journal of Molecular Sciences 25, no. 4: 2064. https://doi.org/10.3390/ijms25042064

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