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. 2026 Jul 10;26:1694. doi: 10.1186/s12870-026-09473-y

CDPK gene family in Camellia nitidissima: genome-wide identification and expression analysis during petal development and hormone treatments

ZiLin Bai 1,2,#, XiaoJuan Xing 2,#, Huaiyuan Wu 3, Lingling Wang 4, Hengfu Yin 1, WeiXin Liu 1,✉
PMCID: PMC13637213  PMID: 42432472

Abstract

Background

Calcium-dependent protein kinases (CDPKs) serve as key sensors and transducers of intracellular calcium signals, playing crucial regulatory roles in diverse physiological processes. However, research on CDPK genes in Camellia nitidissima remains limited.

Results

In this study, we identified 34 CDPK genes distributed across 15 chromosomes in the C. nitidissima genome. Phylogenetic analysis further classified the CDPK gene family into five distinct subfamilies. Promoter cis-element analysis revealed regulatory motifs associated with hormone responses, growth and development, and abiotic stress tolerance. Expression analysis revealed that six CnCDPKs are specifically and highly expressed in petals at the bud stage. In contrast, CnCDPK15/28/31/34 exhibited significantly elevated expression during the senescence phase, suggesting their potential role in petal aging. Eight CnCDPK genes were highly expressed in stamens, with CnCDPK18 showing the highest expression, implicating these genes in stamen development. Collectively, CnCDPK9 and CnCDPK30 responded rapidly to MeJA treatment, whereas CnCDPK3, CnCDPK23, and CnCDPK31 responded rapidly to ET treatment, suggesting that these genes are key regulatory in MeJA- and ET-mediated physiological responses, respectively. Both CnCDPK3 and CnCDPK31 were significantly upregulated by MeJA, ET, and GA₃, suggesting their key roles in hormone signal crosstalk.

Conclusions

Our study identified the CDPK gene family in C. nitidissima, providing new insights into its regulatory roles in floral organ development and hormone signaling.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12870-026-09473-y.

Keywords: Camellia nitidissima, Calcium-dependent protein kinase, Expression analysis, Bioinformatics analysis, Hormone treatment

Background

Calcium-dependent protein kinases (CDPKs) serve as Ca²⁺-activated calcium sensors. By detecting fluctuations in intracellular Ca²⁺ concentrations, CDPKs trigger downstream signaling cascades [1–2]. They also play essential roles in regulating diverse physiological processes in plants, including growth and development, carbon and nitrogen metabolism, cytoskeletal organization, stomatal movement, and responses to biotic and abiotic stresses [3–6].A typical CDPK protein comprises four domains arranged from the N- to C-terminus: an N-terminal variable domain, a Ser/Thr protein kinase domain, a linker domain, and a C-terminal calmodulin-like domain with EF-hand motifs [7–8]. Notably, the EF-hand-containing calmodulin-like domain both binds Ca²⁺ and regulates CDPK catalytic activity. Upon Ca²⁺ binding, this domain undergoes a conformational change that relieves autoinhibition and activates the kinase, thereby enabling its role in signal transduction.

CDPKs are widely distributed across plants, protists, oomycetes, and green algae, forming a large multigene family within diverse plant species [9]. These proteins are involved in diverse plant developmental and physiological processes. Accumulating evidence indicates that CDPKs participate in hormone signaling [10], the regulation of plant growth and development [11], responses to and tolerance of various abiotic and biotic stresses [12], and control of stomatal movement [13]. They are also involved in ion homeostasis, water transport across cellular membranes [14], and defense responses against pathogenic microorganisms [15]. In Oryza sativa, OsCDPK13 and OsCDPK17 are key components of the cold stress signaling pathway [16–19]. Specifically, OsCDPK17 transduces cold stress signals and negatively regulates plant metabolism by suppressing enzymes involved in sugar and nitrogen metabolism [19]. Under high-temperature stress, overexpression of Dioscorea opposita DoCDPK20 in Nicotiana tabacum reduces photosynthetic activity but enhances heat tolerance in the transgenic plants [20]. In Populus euphratica, PeCDPK10 confers both cold and drought tolerance by mediating ABA-induced stomatal closure. Transgenic Arabidopsis thaliana overexpressing PeCDPK10 shows reduced water loss under drought conditions and increased freezing tolerance [21]. In Zea mays, ZmCDPK1regulates the cold stress response. Upon cold exposure, ZmCDPK1 is transcriptionally upregulated and negatively regulates a cold-responsive marker gene, reducing cold tolerance in transgenic A. thaliana [22]. Collectively, these findings indicate that CDPK genes modulate abiotic stress responses in diverse plant species through positive and negative regulatory mechanisms.

Camellia nitidissima is an evergreen shrub in the genus Camellia (family Theaceae), naturally occurring on limestone mountains at 130–460 m elevations [23–24]. First discovered in 1933, it is renowned for its waxy, golden petals [25]. Considered a relict species from the original mountain camellias that survived the Quaternary glaciation, C. nitidissima is often called “living fossil”. Due to its rarity and unique characteristics, it has been called the “panda of the plant world” and the “queen of the Theaceae family ” [26–27], and is officially listed as a rare and endangered protected species in China. Beyond its exceptional ornamental value, C. nitidissima possesses notable medicinal properties. Numerous studies have shown that it is particularly rich in flavonoid compounds [28–30]. Flavonoids, plant‑synthesized natural secondary metabolites, exhibit various biological activities, including antioxidant [31], hypoglycemic [32], anticancer [33–34], and antitumor effects [35–36]. With a long history of use in traditional medicine and as a dietary resource, C. nitidissima is widely employed in folk remedies. In 2010, its leaves were officially approved by China’s Ministry of Health as a novel food resource [37].

CDPK genes have been extensively characterized in various model plant species. However, their regulatory roles in C. nitidissima remain poorly understood. To elucidate the potential functions of CDPK genes in C. nitidissima, we performed a genome-wide identification and expression analysis of the CDPK gene family. Our findings provide a foundation for future functional studies to elucidate the roles of CDPKs in the development and stress responses of C. nitidissima.

Results

Identification, physicochemical characterization, and subcellular localization prediction of CnCDPKs

Using A. thaliana AtCDPKs as references, we performed a BLASTP search with TBtools. Candidate sequences were filtered using Pfam and CDD, only those containing the CDPK hallmark domains (Ser/Thr kinase domain and EF-hand Ca²⁺-binding domain) were retained. This yielded 34 CDPK family members in C. nitidissima, which were named CnCDPK1–CnCDPK34 based on their chromosomal locations. Physicochemical analysis showed that these proteins range from 505 to 621 amino acids in length, with molecular weights of 56.47–69.66 kDa (average 62.01 kDa) and isoelectric points (pI) of 5.18–9.16. Most are acidic (pI < 7), and only seven are basic (pI > 7). The instability index ranges from 32.18 to 50.44, and the aliphatic index from 76.18 to 90.60, indicating that all CnCDPKs are hydrophilicity and mostly stable. Subcellular localization predictions revealed diverse patterns: 16 CnCDPKs in the cytoplasm, 7 in chloroplasts, 6 in the nucleus, 2 in mitochondria, 2 in the endoplasmic reticulum, and 1 in peroxisomes (Table S1).

Chromosomal mapping of CDPK gene family in C. nitidissima

Chromosomal localization analysis showed that the 34 CnCDPK genes are distributed across 15 chromosomes, with chromosome 12 harboring the most (six: CnCDPK26–CnCDPK31), followed by chromosome 1 (four: CnCDPK1–CnCDPK4). Chromosomes 2, 3, and 6 each carry three genes: CnCDPK5–CnCDPK7 on chr2, CnCDPK8–CnCDPK10 on chr3, and CnCDPK14–CnCDPK16 on chr6. Chromosomes 5, 8, 9, 10, and 11 each harbor two CDPK genes. The remaining five chromosomes each harbor one gene: CnCDPK11 on chr4, CnCDPK17 on chr7, CnCDPK32 on chr13, CnCDPK33 on chr14, and CnCDPK34 on chr15 (Fig. 1).

Fig. 1.

Fig. 1

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Chromosomal distribution of CDPK family in C. nitidissima

Phylogenetic analysis of the CDPK gene family in C. nitidissima

To investigate the phylogenetic relationships of the CDPK gene family in C. nitidissima, we performed a phylogenetic analysis using MEGA, incorporating 98 protein sequences from A. thaliana (34), O. sativa (30), and C. nitidissima (34). The CnCDPK family is divided into five distinct subfamilies (I–V) (Fig. 2), ranging from 1 to 10: Subfamily III has the most (10 members), followed by Subfamilies I and II (8 each), Subfamily IV (7), and Subfamily V (only 1). Notably, in Subfamilies II and V, CnCDPK proteins cluster closely with A. thaliana homologs on short, well-supported branches, indicating high sequence homology. This suggests that the CDPK gene family in C. nitidissima is phylogenetically closer to A. thaliana than to O. sativa.

Fig. 2.

Fig. 2

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The evolutionary tree of CDPK genes from Camellia nitidissima, Arabidopsis thaliana and Oryza sativa

Collinearity analysis of the CDPK gene family in C. nitidissima

Intraspecific collinearity analysis revealed frequent gene duplication events among CDPK genes in C. nitidissima.,with 21 collinear gene pairs identified among the 34 genes, indicating that duplication is a major driver of CDPK family expansion in this species (Fig. 3A).

Fig. 3.

Fig. 3

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Intraspecies collinearity of Camellia nitidissima (A), Collinear analysis of CnCDPKs from Camellia nitidissima with Arabidopsis thaliana and Oryza sativa (B)

Comparative genomic synteny analysis was performed between C. nitidissima, the dicot A. thaliana, and the monocot O. sativa. The results showed significantly higher synteny with A. thaliana (52 homologous pairs) than with O. sativa (17 pairs), supporting a closer phylogenetic relationship between C. nitidissima and A. thaliana (Fig. 3B). Notably, CnCDPK10 exhibited three collinear relationships with A. thaliana and two with O. sativa, suggesting a conserved and potentially important role in the evolution of CDPK-mediated signaling across monocots and dicots.

Genetic structure and motif analysis of the CDPK gene family in C. nitidissima

Conserved motifs critical for protein structure and function, were analyzed using the MEME suite. We identified 10 distinct motif types among CnCDPK proteins, with each protein containing 8–10 motifs. Proteins within the same phylogenetic clade shared greater number of conserved motifs and also exhibited higher sequence similarity (Fig. 4B). Domain architecture analysis revealed that all 34 CnCDPK proteins exhibit high sequence conservation and possess either the CaMK + PTZ or CaMK + FRQ domain configuration, confirming their classification as canonical CDPKs (Fig. 4C). Gene structure analysis showed that all CnCDPK genes except CnCDPK2 are shorter than 20 kb; CnCDPK2 is the longest and CnCDPK6 the shortest. Additionally, 16 genes (47% of the family) share a conserved eight‑exon, seven‑intron structure (Fig. 4D).

Fig. 4.

Fig. 4

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Phylogenetic tree and Gene structure of CDPK family in Camellia nitidissima. A: Phylogenetic Tree of the CnCDPK Gene Family; B: Analysis of the conserved motifs; C: conservative structure domains; D: gene structure

Prediction of cis-acting elements in the promoter regions of the CDPK gene family in C. nitidissima

To investigate the functions of the CDPK family in C. nitidissima, we analyzed cis-acting elements in the 2000-bp promoter of CnCDPK genes. These elements were primarily grouped into three categories: growth and development, hormone and abiotic stress (Fig. 5).

Fig. 5.

Fig. 5

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Analysis of cis-elements in CnCDPKs 2000 bp upstream promoter

Growth and development-related elements: Eight types were identified, with ight-responsive elements being the most abundant (49 occurrences), followed by meristem-related (9), flavonoid biosynthesis (4), circadian control (3), palisade epidermis differentiation (3), cell cycle regulation (3), seed-specific regulation (3), and wound response (2).

Hormone-responsive elements: Five types were found. Methyl jasmonate (MeJA)-responsive elements were most frequent (80), followed by abscisic acid (ABA)-responsive (36), salicylic acid (SA)-responsive (20), gibberellin (GA)-responsive (16), and auxin-responsive (3). Their distribution across subfamilieswas roughly proportional to subfamily size.

Stress-responsive elements: Five types related to adverse conditions were predicted. These included anaerobic induction (79), cold response (19), drought response (18), defense and stress responses (16), and hypoxia-specific induction (8).

Overall, these findings suggest that CnCDPK genes play important roles in the growth, development and abiotic stress responses of C. nitidissima.

Analysis of expression patterns of CDPK gene family in C. nitidissima

Tissue-specific expression analysis of CnCDPK genes were performed in sepals, petals, stamens, pistils, leaves, roots, and fruits(Fig. 6A). CnCDPK3 and CnCDPK11 were highly expressed across all tissues, with CnCDPK3 highest in fruit. Eight genes (CnCDPK6/18/19/24/25/26/27/31) were markedly upregulated in stamens, among which CnCDPK18 exhibited the strongest expression. CnCDPK9 was notably upregulated in both petals and stamens; CnCDPK22 was highly expressed in stamens and roots. CnCDPK16 was preferentially expressed in leaves and fruits, while CnCDPK14 and CnCDPK15 were predominantly root-specific.

Fig. 6.

Fig. 6

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Expression characteristics of CnCDPKs gene in different tissue parts (A), Expression characteristics of CnCDPKs gene in different flower development stages (B) in Camellia nitidissima. Se: Sepal; Pe: Petal; St: Stamen; Pi: Pistil; L: Leaf; R: Root; Fr: fruit

To investigate potential roles in petal development, we examined expression of all 34 CnCDPK genes across six sequential stages in C. nitidissima petals (Fig. 6B). The heatmap revealed substantial variation across stages. CnCDPK3, CnCDPK11, CnCDPK21, CnCDPK22, and CnCDPK33 were consistently highly expressed throughout all stages, suggesting housekeeping or broadly regulatory roles. CnCDPK7, CnCDPK13, CnCDPK15, CnCDPK23, CnCDPK30, and CnCDPK32 were upregulated during early bud stages (S0–S2), implicating their involvement in early petal formation or differentiation. Notably, CnCDPK15 also showed pronounced expression at the senescence stage (S5), suggesting a dual role. CnCDPK28, CnCDPK31, and CnCDPK34 were specifically and highly expressed at S5, strongly suggesting involvement in petal senescence. In contrast, CnCDPK9 was predominantly expressed at the earliest stage (S0), indicating a potential role in petal initiation or patterning. These dynamic expression profiles indicate that individual CnCDPK genes likely execute specialized functions at distinctdevelopmental time points, underscoring the necessity for further functional characterization.

Expression patterns of CnCDPKs in C. nitidissima under MeJA treatment

As shown in Fig. 7, among the 11 CnCDPK genes examined following MeJA treatment, four (CnCDPK3, CnCDPK23, CnCDPK31, and CnCDPK32) were significantly upregulated. CnCDPK3, CnCDPK23, and CnCDPK31 responded rapidly (within 3 h), whereas CnCDPK32 responded more slowly (after 12 h). CnCDPK3, CnCDPK23, and CnCDPK32 reached peak expression in the late phase (48–72 h), whereas CnCDPK31 showed high expression in both early (3–6 h) and late (72 h) phases.

Fig. 7.

Fig. 7

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Expressions characteristics of CnCDPKs genes in MeJA hormone treatments

Other genes exhibited more dynamic patterns. CnCDPK9 and CnCDPK30 showed initial upregulationwithin3–48 h, peaking at 3 h and 12 h, respectively. Both genes were downregulation at 72 h. CnCDPK7, CnCDPK11, and CnCDPK33 showed initial downregulation (3–24 h) followed by upregulation at 48–72 h, peaking at 72 h. CnCDPK16 exhibited an up-down-up pattern: up at 3 h, down at 6–24 h, and up again at 48–72 h. CnCDPK12 exhibited a complex, wave-like expression pattern with alternating up‑ and downregulation (down‑up‑down‑up), showing significant upregulation at 6 h and 48 h and downregulation at other time points.

These results suggest that CnCDPK9 and CnCDPK30 may act as rapid signal sensors and initiators of downstream defense programs in MeJA-mediated responses, thereby acting as key regulatory factors of stress responses. CnCDPK31 participates in both the early perception of MeJA and late-stage amplification of physiological responses. CnCDPK12 and CnCDPK16 may be early-response genes induced by MeJA. Conversely, which peaked exclusively in the late phase, may play key roles in the late-stage regulation of MeJA-mediated physiological responses.

Expression patterns of CnCDPKs in C. nitidissima under ET treatment

As shown in Fig. 8, following ET treatment, three CnCDPK genes (CnCDPK3, CnCDPK23, and CnCDPK31), which responded rapidly (within 3 h) and peaked at 24 h.

Fig. 8.

Fig. 8

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Expressions characteristics of CnCDPKs genes in ET hormone treatments

Other genes showed distinct dynamic patterns. CnCDPK7, CnCDPK9, CnCDPK30, and CnCDPK33 exhibited initial downregulation followed by upregulation. Specifically, CnCDPK7 and CnCDPK9 were significantly downregulated at 12 h and 6 h, respectively, followed by upregulation from 24 to 72 h, with peak expression at 24 h. CnCDPK32 showed initial upregulation (3–48 h) followed by downregulation at 72 h. CnCDPK16 displayed a down-up-down pattern: down at 6–12 h, up at 24–48 h, and down again at 72 h. CnCDPK11 and CnCDPK12 exhibited an up-down-up pattern. Specifically, CnCDPK11 was upregulated at 3 h, downregulated at 6 h, and upregulated again from 12 to 72 h; CnCDPK12 was upregulated at 3 h, downregulated at 6–12 h, and upregulated again from 24 to 72 h.

These results indicate that CnCDPK3, CnCDPK23, and CnCDPK31 can rapidly sense and respond to ethylene stress, thereby playing important roles in ET-mediated physiological responses. CnCDPK7, CnCDPK9, CnCDPK16, CnCDPK30, and CnCDPK33 may be ET-induced late-response genes, whereas CnCDPK11 and CnCDPK12 respond to ET signaling in both early and late phases.

Expression patterns of CnCDPKs in C. nitidissima under GA3 treatment

As shown in Fig. 9, following GA₃ treatment, six of the 11 CnCDPK genes (CnCDPK3, CnCDPK7, CnCDPK9, CnCDPK16, CnCDPK23, and CnCDPK31) showed sustained and significant upregulation. These genes responded rapidly (within 3 h) and peaked at either 6–72 h.

Fig. 9.

Fig. 9

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Expressions characteristics of CnCDPKs genes in GA3 hormone treatments

Other genes exhibited an up-down-up pattern. CnCDPK11 was upregulated at3–24 h, downregulated at 48 h, and upregulated again at 72 h. The other four genes (CnCDPK12, CnCDPK30, CnCDPK32, and CnCDPK33) were upregulated at 3–6 h, downregulated at 12 h, and upregulated again from 24 to 72 h.

Overall, these results demonstrate that GA₃ treatment significantly upregulated CnCDPK genes expression, suggesting that they play crucial roles in gibberellin biosynthesis and signaling pathways in C. nitidissima.

Discussion

Plants are continuously exposed to diverse environmental stimuli throughout their life cycle. To cope with these challenges, they have evolved sophisticated mechanisms that enable to perceive external signals and modulate physiological responses accordingly. These mechanisms typically involve stimulus perceptionand signal transduction, which ultimately triggers a cascade of chemical, molecular, biochemical, and physiological changes that enhance stress resilience. CDPKs represent a key class of calcium-regulated, calcium-sensing protein kinases that serve a central role in converting calcium signals into adaptive cellular responses. They function as crucial signaling hubs in plants, mediating responses to biotic and abiotic stresses, immune activation, hormone biosynthesis and signaling, and various aspects of growth and development [38].

In this study, bioinformatics analysis identified34 CnCDPK family genes in C. nitidissima, a number slightly higher than that reported in O. sativa. This discrepancy may reflect varying degrees of CDPK gene expansion across different plant species. Physicochemical analysis revealed that CnCDPKs had negative GRAVY values, indicating they were hydrophilic proteins. This is consistent with finding in yellowhorn [39]; XsCDPKs are also hydrophilic proteins, and CsCDPK17 from the tea plant [40] is similarly classified as a hydrophilic protein. The pI of most CnCDPK proteins is less than 7, consistent with findings from studies on yellowhorn [39], C. sinensis [40, 41], P. mume [42], and Rosa chinensis [43]. Furthermore, the ranges of other physicochemical properties are also similar. Subcellular localization predictions indicate that C. nitidissima CDPKs are distributed across multiple compartments, including the cytoplasm, nucleus, endoplasmic reticulum, mitochondria, chloroplasts, and peroxisomes. This pattern is consistent with findings in A. thaliana [44], where CDPKs are predominantly localized to the cytoplasm, and with the cytoplasmic enrichment observed in Rosa roxburghii [45]. Furthermore, in C. nitidissima, 47% of CnCDPK proteins are predicted to localize to the cytoplasm, further supporting this conserved subcellular distribution.

Research indicates that CDPK genes in various plant species are closely linked to antioxidant enzymes [46], which are widespread in plants and participate in multiple metabolic pathways. Overexpression of a CDPK gene in Zea mays enhances superoxide dismutase and peroxidase activities [47], thereby accelerating plant metabolic. In C. nitidissima, CnCDPK32 localizes to peroxisomes and may regulate peroxidase activity. Most CnCDPKs genes consist of 8 exons and 7 introns. Studies on yellowhorn [39], Camellia sinensis [40, 41], P. mume [42], and R. chinensis [43] have also found that these genes typically contain 7–8 exons. The similar exons and introns numbers suggest that they may share common patterns of genetic evolution. All C. nitidissima CDPKs contain either the CaMK + PTZ or CaMK + FRQ domain combination, consistent with observations in apple (Malus domestica) [48]. The number, types, and spatial arrangement of motifs within the CnCDPK family are largely conserved, although variations exist among subfamilies. This diverse motif distribution suggests that these CDPKs may have undergone functional diversification in C. nitidissima [49].

Gene duplication events are a major driver of gene family expansion [50]. Intraspecific collinearity analysis revealed 21 collinear gene pairs among C. nitidissima CDPKs, suggesting that most CnCDPK genes likely arose from recent duplication rather than being retained from ancestral genes. Interspecific collinearity analyses among C. nitidissima, O. sativa, and A. thaliana identified homologous CnCDPK genes in O. sativa and A. thaliana. In plants, the CDPK gene family regulates growth and development and plays critical roles in hormone signaling transduction, responses to abiotic stresses, and innate immune responses [4–6].

Cis-acting elements analysis of the CnCDPK gene family revealed that their promoters harbor numerous hormone-responsive, stress-responsive, and growth-related regulatory elements. Among these, the elements responsive to methyl jasmonate (MeJA) and abscisic acid (ABA) are the most abundant. MeJA is a key plant hormone, regulates flowering, fruiting, and senescence [51], and its exogenous application triggers plant defense responses [52]. ABAis a central hormone in plant stress responses [53], Studies on Schisandra yellowhorn [39], C. sinensis [40, 41], and R. chinensis [43] have also shown that CDPKs commonly contain both the methyl jasmonate (MeJA) response element and the abscisic acid (ABA) response element. Further supporting the hypothesis that the CDPK gene family plays a role in stress response and signal transduction pathways in C. nitidissima. Recent studies by Martins et al. [54]. reported that calcium significantly influences flavonoid production, particularly by enhancing the accumulation of quercetin-type flavonoids. Additionally, exogenous calcium increase flavonoid levels in peanuts [55]. Flavonoid metabolism‑related elements were also identified in the CsCDPK17 promoter of C. sinensis [40]. As important secondary metabolites of C. nitidissima, flavonoids contribute to its distinctive golden-yellow petals coloration and possess significant physiological and pharmacological value. Notably, the promoters of CnCDPK7, CnCDPK24, CnCDPK26, and CnCDPK27 contain MYB-binding sites implicated inflavonoid biosynthesis genes regulation. These findings suggest that CnCDPK7, CnCDPK24, CnCDPK26, and CnCDPK27 may play a role in regulating flavonoid biosynthesis in C. nitidissima, potentially through Ca²⁺-mediated signaling pathways. This provides candidate genes and a research foundation for future efforts to enhance the flavonoid content and nutritional value of C. nitidissima through molecular breeding.

Expression pattern analysis revealed that CnCDPK15, CnCDPK28, CnCDPK31, and CnCDPK34 were significantly upregulated during the petal senescence stage in C. nitidissima (S5). Similarly, in Carthamus tinctorius, CtCDPK25 expression was markedly higher at the flower decay stage than at other developmental stages [56], suggesting that these four CnCDPK genes may play a role in regulating petal senescence. In contrast, CnCDPK3, CnCDPK11, CnCDPK21, CnCDPK22, and CnCDPK33 were consistently highly expressed across all petal developmental stages, suggesting they likely function in general growth and development. CDPKs are widely distributed in plant cells, tissues, and organs [57–59]. In C. nitidissima, tissue-specific expression analysis revealed that some genes were expressed in all tissues but at varying levels. For instance, CnCDPK22 was highly expressed in stamens and roots; CnCDPK16 showed higher expression in leaves and fruits than in other tissues, and CnCDPK14 and CnCDPK15 were strongly expressed in roots. Similar patterns have been observed in Vicia faba, where VfCDPK1 was detected in all tissues but was notably more abundant in leaves and lower epidermis than in roots, stems, and leaf mesophyll [60]. PmCDPK3/6 is highly expressed in roots, while PmCDPK7 is highly expressed in leaves [42]. Some CDPKs exhibit organ- or tissue-specific expression; for instance, BnCDPK1 is predominantly expressed in Brassica napus leaves [61]. CsCDPK17 is specifically expressed in fruits [40]. In contrast, CnCDPK6, CnCDPK18, CnCDPK19, CnCDPK24, CnCDPK25, CnCDPK26, CnCDPK27, and CnCDPK31 were specifically expressed in stamens, RcCDPK8/11/13 is also specifically expressed in stamens [43]. This suggests their potential involvement in stamen development. These tissue-specific expression differences imply that individual CnCDPK genes may differentially regulate growth and development across various plant tissues. Together, these findings provide a foundation for future functional characterization of CDPKs in C. nitidissima and offer new insights into the functional diversity of this gene family.

Numerous studies have revealed that CDPK interacts with plant hormones in defense and development processes [62]. Earlier studies have shown that CDPK regulation is a Ca²⁺-dependent response triggered by plant hormones. Changes in hormone levels alter intracellular Ca²⁺ concentrations, and CDPKs, as Ca²⁺ sensors, transmit hormone signals to downstream pathways [63]. In Arabidopsis thaliana, CDPK28 is a key MeJA regulator [64]. Following MeJA treatment, TaCDPK17 expression remained at high levels between 1 and 12 h [65]. NtCDPK4 and NtCDPK5 respond to MeJA signaling and influence its early biosynthesis [66]. In this study, CnCDPK9 and CnCDPK30 rapidly respond to MeJA signals; CDPK31 participates in early MeJA signal transduction and was also significantly upregulated in the late phase; CnCDPK12 and CnCDPK16 respond to MeJA induction in the early phase; while other genes exhibit significant changes in the late phase of MeJA induction. This indicates that CnCDPKs play a key role in regulation at different stages.

CDPK is also involved in the regulation and transmission of ethylene signals [67]. The phosphorylation level of HbCDPK changed significantly following ethylene treatment [68]. StCDPK5 can be induced by ethylene and plays a key role in ethylene-mediated flower stalk abscission in Solanum lycopersicum [69]. In this study, the expression levels of CnCDPK3, CnCDPK23, and CnCDPK31 were also upregulated by ET. GA3 significantly upregulates CnCDPK expression, consistent with other studies showing CDPK activity was higher in seeds treated with gibberellin than in untreated seeds [70]. StCDPK1 mediates gibberellin signaling and promotes tuber formation [71]. Exogenous gibberellin upregulated OsCDPK13 [72], NtCPK4 [73], and IiCDPK2 [74]. This suggests that the GA3 hormone likely regulates downstream physiological responses by modulating the calcium signaling network within C. nitidissima cells (Upgrade CnCDPKs).

Under different treatment conditions, CnCDPKs participate in various regulatory pathways via distinct expression patterns perform their biological functions. The CnCDPKs genes respond to MeJA, ET, and GA3 treatments to varying degrees, indicating that the response patterns differ under different stress conditions. It is worth noting that CnCDPK3/31 responds simultaneously to MeJA, ET, and GA3, suggesting that it may serve as a downstream convergence point for these three signaling pathways, responsible for integrating different hormonal signals and participating in the various growth and developmental processes they mediate. CnCDPK7/33 exhibits the same expression pattern following MeJA and ET treatment, while CnCDPK12 shows the same pattern following treatment with GA3 and ET. This suggests that they may serve as common response elements in the MeJA-ET and GA3-ET pathways, playing synergistic or overlapping roles. In contrast, CnCDPK9/16/30 exhibited opposite expression patterns following MeJA and ET treatment, suggesting that it may serve as a key node in the crosstalk between these pathways, playing opposing roles.

CnCDPK3 is highly expressed in various tissues of C. nitidissima and across different developmental stages of its petals, and is significantly induced by MeJA. Previous studies have shown that MeJA can regulate both vegetative and reproductive growth in plants [75]. These suggests that CnCDPK3 may play an important role in the MeJA-mediated pathways regulating plant growth and development as well as petal development; In contrast, CnCDPK31 exhibits specific expression in the stamens of C. nitidissima. CnCDPK31 is significantly upregulated by GA3. The mechanisms by which gibberellins regulate stamen development and filament elongation have been previously reported [76], it is plausible that CnCDPK31 may be involved in the gibberellin-mediated regulatory pathway of stamen development; Furthermore, CnCDPK31 is specifically expressed during the S5 of C. nitidissima petal development, and it is significantly induced by ethylene. Studies on ethylene have shown that it promotes petal senescence and the floral organs abscission [76], suggesting that CnCDPK31 may be involved in the regulatory pathways of ethylene-mediated petal senescence. C. nitidissima is renowned for its unique golden-yellow flower color. Our results indicate that CnCDPK3 and CnCDPK31 may play important roles in flower development, providing new targets for future molecular breeding and germplasm innovation in this species. However, this study only analyzed gene expression patterns, their functions and regulatory mechanisms require further investigation.

Conclusions

In this study, we conducted genome-wide identification, phylogenetic, structural, and expression analyses of the CDPK gene family in C. nitidissima. We identified 34 CnCDPK genes, classified them into five subfamilies unevenly distributed across 15 chromosomes. Gene duplication and collinearity analyses indicated that segmental duplication greatly contributed to the expansion and evolutionary conservation of the CnCDPK family. Promoter cis-element analysis revealed abundant elements associated with hormone responses, growth and development, and abiotic stress, implying diverse regulatory potentials of CnCDPKs. Expression profiling revealed that CnCDPK genes exhibited distinct tissue-specific and petal development-stage-specific patterns, with several members strongly induced during petal senescence or stamen development. Furthermore, RT-qPCR confirmed that multiple CnCDPKs rapidly and significantly to MeJA, ET, and GA₃ treatments. Among them, CnCDPK9/30 were preferentially sensitive to MeJA, CnCDPK3/23/31 were rapidly responsive to ET, and most CnCDPKs were strongly upregulated by GA₃. Notably, CnCDPK3 and CnCDPK31 responded to all three hormones, suggesting core roles in hormone crosstalk. Collectively, these findings show that CnCDPKs are widely involved in regulating petal development, stamen formation, and hormone signaling pathways in C. nitidissima. Further functional validation can be conducted to elucidate the complete hormonal signaling pathway, clarify the biological functions of CnCDPK3/31 in petal development, stamen development, and petal senescence, and investigate whether CnCDPKs regulate flavonoid biosynthesis genes by phosphorylating MYB transcription factors. This research could also provide molecular breeding targets for improving flower color, extending flowering periods, and enhancing pharmacological components in C. nitidissima.

Materials and methods

Plants Materials

The plant materials used in this study were approximately 15-year-old C. nitidissima individuals from the Camellia germplasm repository at the Subtropical Forestry Research Institute, Chinese Academy of Forestry, Hangzhou, Zhejiang, China. Samples were collected from six distinct developmental stages of C. nitidissima petals: bud stage (S0), mid-bud stage (S1), late-bud stage (S2), half-open stage (S3), full-bloom stage (S4), and senescence stage (S5). S0: the petals were translucent, and the buds were tightly closed; S1: the petals were light green, and the buds had swollen compared to the S0 stage but remained tightly closed; S2: the buds were loosely open; S3: The flower buds had opened, the petals were yellow, and the stamens were clearly visible; S4: The petals were fully open, yellow in color, and showed little change from the S3 stage; S5: The petals began to shrivel. Flowers at the appropriate stage were collected between 9:00 and 10:00 a.m. Each sample comprised pooled petals from three plants, with 10–20 flowers taken per plant. Three independent biological replicates were set up for each developmental stage. Immediately after collection, all samples were flash-frozen in liquid nitrogen and stored at − 80 °C in an ultra-low-temperature freezer until further analysis.

Identification of CDPK genes family in C. nitidissima

The genome assembly and GFF annotation files for C. nitidissima were obtained from the Genome Warehouse (GWH) at the National Genomics Data Center (https://ngdc.cncb.ac.cn/gwh/)under accession number GWHFILD00000000.1. To identify CDPK family members, we retrieved 34 A. thaliana CDPK protein sequences and 30 O. sativa CDPK protein sequences from the literature and the Ensembl Plants database (https://plants.ensembl.org/index.html). Using the A. thaliana CDPK proteins as queries, we performed local BLASTP searches against the C. nitidissima proteome using TBtools, retaining the best-matching hits based on alignment quality and E-value thresholds (E-value less than 1e-5). Candidate sequences were then validated for the hallmark CDPK domains using the Pfam database (https://pfam.xfam.org/ ): the Ser/Thr protein kinase domain (Pfam ID: PF00069) and the EF-hand domain (Pfam ID: PF13499). Only sequences containing both the Pkinase and EF-hand domains were retained, duplicates and sequences lacking either domain were excluded. The identified CnCDPK protein sequences were analyzed for domain architecture using the Conserved Domain Database (CDD) tool on NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) to determine whether they harbored the CaMK + PTZ or the CaMK + FRQ configuration. Finally, the CDPK-specific entry (IPR050205) in the InterPro database was used to verify inclusion, thereby confirming their classification within the CDPK family.

Physicochemical characterization and chromosomal localization of the CDPK gene family in C. nitidissima

We analyzed the physicochemical properties proteins(amino acid length, molecular weight, and isoelectric point)of C. nitidissima CDPK using the ExPASy ProtParam tool (https://www.expasy.org). Subcellular localization was predicted using WoLF PSORT online platform (https://wolfpsort.hgc.jp/). CnCDPK Chromosomal positions were extracted from the C. nitidissima genome GFF file and visualized with TBtools.

Phylogenetic analysis of the CDPK gene family in C. nitidissima

Phylogenetic trees of the CDPK families from A. thaliana, O. sativa, and C. nitidissima were constructed using the Neighbor-Joining (NJ) method in MEGA. Bootstrap analysis was performed with 1,000 replicates, and other parameters were set to default. The resulting trees were subsequently visualized and refined using the iTOL online platform (https://itol.embl.de/).

Collinearity analysis of the CDPK gene family in C. nitidissima

The genome assembly and annotation files for C. nitidissima were downloaded from the Genome Warehouse (GWH) at the National Genomics Data Center (https://ngdc.cncb.ac.cn/gwh/). Genome and annotation data for O. sativa and A. thaliana were obtained from the Ensembl Plants database (https://plants.ensembl.org/index.html). Chromosomal locations of the CDPK genes were analyzed and visualized with TBtools. Intraspecific and interspecific collinearity relationships were identified using the OneCnepMCScanX plugin integrated within the TBtools suite.

Genetic structure and motif analysis of the CDPK gene family in C. nitidissima

Conserved domain analysis and domain architectures generation were performed using the NCBI Conserved Domain Search (CD-Search) tool. Gene structure analysis was carried out using the Gene Structure Display Server, and identified conserved motifs in C. nitidissima CDPK using the MEME Suite web server (https://meme-suite.org/meme/). The number of predicted motifs was set to 10, with other parameters at default. Gene structure and motif composition were visualized using TBtools.

Prediction of cis-acting elements in the promoter regions of the CDPK gene family in C. nitidissima

We extracted a 2,000-bp genomic sequence upstream of the start codon of each CDPK gene as the putative promoter region using TBtools. Cis-acting elements within these promoter were predicted using the PlantCARE online tool (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/). We then imported the prediction results into TBtools for visualization and further analysis.

Analysis of expression patterns of CDPK gene family members in C. nitidissima

Transcriptome data for C. nitidissima were retrieved from NCBI SRA (BioProject: PRJNA909942). Sequence identifiers for the 34 CnCDPK genes were obtained via sequence alignment and annotation. The expression levels (FPKM) were processed and analyzed. FPKM values were log₂-transformed for normalization, and expression patterns were visualized using TBtools.

Analysis of CDPK gene expression in C. nitidissima under different hormone treatments

Since the promoter regions of CnCDPKs genes contain various hormone response elements, C. nitidissima leaves were sprayed with ET (1 mmol/L), MeJA (100 µmol/L), and GA3 (1 mmol/L) as described previously [77, 78]. C. nitidissima leaves were collected at various time points (0, 3, 6, 12, 24, 48, and 72 h) and stored in a -80℃ ultra-low temperature freezer. After extracting total RNA from the samples using the kit, reverse transcription was performed to synthesize cDNA and store it at -20℃ for later use. Based on transcriptome data from different tissues and flower development stages of C. nitidissima (PRJNA909942), 11 genes with relatively high expression levels were selected for qPCR analysis under hormone treatment, and specific primers for CnCDPKs were designed using Primer Premier v6.0 software; the primer sequences are listed in Table S2.RT-qPCR was performed with three biological replicates using GAPDH as the reference gene. Relative expression levels of the CnCDPKs gene were calculated using the 2−△△Ct method. Statistical analyses were performed using Excel, GraphPad Prism v6.01, and SPSS v22.0.

Supplementary Information

12870_2026_9473_MOESM1_ESM.docx (54.8KB, docx)

Supplementary Material 1. Table S1. Characterization of the CDPK gene family members of Camellia nitidissima.Table S2. Primers for RT-qPCR.

Authors’ contributions

Z.B. and X.X.: Investigation, Formal Analysis, Writing—original draft, H.W. and H.Y.: Writing—review and editing. L.W.: Visualization. W.L.: Conceptualization, Formal Analysis, Writing—review and editing. All the authors reviewed and approved the final manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by National Natural Science Foundation of China (32371842) and Fundamental Research Funds of CAF (CAFYBB2023MB005).

Data availability

All the data presented in this study are available in the manuscript and its additional files.

Declarations

Ethics approval and consent to participate

This article does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

ZiLin Bai and XiaoJuan Xing contributed equally to this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

12870_2026_9473_MOESM1_ESM.docx (54.8KB, docx)

Supplementary Material 1. Table S1. Characterization of the CDPK gene family members of Camellia nitidissima.Table S2. Primers for RT-qPCR.

Data Availability Statement

All the data presented in this study are available in the manuscript and its additional files.

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