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International Journal of Molecular Sciences logoLink to International Journal of Molecular Sciences
. 2025 Dec 31;27(1):431. doi: 10.3390/ijms27010431

Genome-Wide Identification and Analysis of bZIP Transcription Factors in Coptis chinensis Reveals Their Regulatory Roles in Stress Responses

Wuke Wei 1,2,3, Zijian Le 3,4, Lianan Guo 1, Rangyu Mo 1, Yu Wang 1,*, Yuan Pan 1,*
Editor: Martin Bartas
PMCID: PMC12786017  PMID: 41516304

Abstract

The basic leucine zipper (bZIP) transcription factors play crucial roles in plant growth and stress adaptation. However, a comprehensive genome-wide analysis of this family has been lacking in the medicinal plant Coptis chinensis. In this study, we identified 55 bZIP genes (CcbZIPs) from the C. chinensis genome and systematically classified them into 12 subfamilies through phylogenetic analysis with Arabidopsis thaliana. Notably, subfamilies L and O were absent, while two orphan genes were discovered, indicating lineage-specific evolution. Expression profiling revealed that numerous CcbZIPs respond dynamically to temperature and light stresses in a tissue-specific manner. These findings provide a foundation for understanding the regulatory roles of CcbZIPs in environmental adaptation and secondary metabolism, offering potential genetic targets for future breeding aimed at improving stress tolerance and medicinal compound production in C. chinensis.

Keywords: Coptis chinensis, bZIP transcription factors, genome-wide analysis, abiotic stress, gene expression

1. Introduction

Transcription factors (TFs) are central regulators of gene expression, governing diverse processes in plant growth, development, and adaptation to environmental stresses [1,2]. Among various TF families, the basic leucine zipper (bZIP) family represents one of the largest and most evolutionarily conserved groups in eukaryotes [3]. Typical bZIP proteins are characterized by two functional domains: an N-terminal basic region that facilitates sequence-specific DNA binding, and a C-terminal leucine zipper domain responsible for dimerization [4]. This structure enables bZIP TFs to recognize a variety of DNA motifs—such as G-box (CACGTG), A-box (TACGTA), and C-box (GACGTC)—thereby regulating distinct transcriptional programs involved in numerous signaling pathways [1,5]. Functionally, bZIPs participate in a wide spectrum of physiological events, including embryogenesis, photomorphogenesis, flowering time control, and responses to biotic and abiotic stresses, underscoring their broad regulatory significance [6,7].

Genome-wide studies have systematically identified bZIP members across numerous plant species, revealing both conserved and lineage-specific evolutionary patterns [8]. In the model plant Arabidopsis thaliana, 78 bZIP genes have been classified into 13 subfamilies, with many members implicated in abscisic acid (ABA) signaling, pathogen defense, and energy homeostasis [1]. Similarly, in horticultural and medicinal plants such as litchi [9], wheat [10], rice [11], maize [12], tomato [13], potato [14], banana [15], cucumber [16], sesame [17], pomegranate [18], etc. bZIP TFs have been linked to fruit senescence, stress tolerance, and the biosynthesis of specialized metabolites like terpenoids [19]. Notably, bZIPs are increasingly recognized as key regulators of secondary metabolism in medicinal plants. For instance, AabZIP1, AabZIP9, and AaTGA6 in Artemisia annua positively regulate the biosynthesis of the antimalarial compound artemisinin [20,21,22], while bZIPs in Carthamus tinctorius modulate flavonoid biosynthesis [23]. These findings highlight the potential of engineering bZIP TFs to enhance the production of valuable phytochemicals [24].

As integrators of environmental signals, bZIP TFs also play pivotal roles in light and temperature response pathways. They participate in photomorphogenesis through HY5-mediated signaling and in temperature stress responses via both ABA-dependent and independent mechanisms [25]. Examples include MdHY5, which regulates light-induced anthocyanin biosynthesis in apple, and OsbZIP52, which enhances cold tolerance in rice [19,26]. These studies position bZIP TFs as critical hubs within stress-responsive networks, linking environmental perception to metabolic reprogramming [27].

Coptis chinensis (Chinese goldthread), a perennial herb of the Ranunculaceae family, is a renowned medicinal plant whose rhizomes accumulate bioactive isoquinoline alkaloids—such as berberine, palmatine, and coptisine—which exhibit antimicrobial, anti-inflammatory, and anticancer properties [28]. Environmental factors, particularly light and temperature, are known to influence both growth and alkaloid accumulation in medicinal plants [29,30]. However, despite its medicinal significance, the molecular mechanisms governing the biosynthesis and regulation of these valuable compounds remain poorly characterized. Moreover, how abiotic signals are perceived and transduced into transcriptional reprogramming—specifically through bZIP TFs—has not been systematically investigated in C. chinensis.

Therefore, to bridge this knowledge gap, we performed a genome-wide identification and analysis of the bZIP family in C. chinensis. This study aims to characterize the phylogenetic relationships, gene structures, conserved motifs, and chromosomal distributions of CcbZIP genes. Furthermore, we analyzed their expression patterns under different temperature and light conditions. Our findings provide a foundational resource for understanding the regulatory roles of CcbZIP TFs in the environmental adaptation and secondary metabolism of this medically important species.

2. Results

2.1. Genome-Wide Identification, Classification and Phylogenetic Analysis of CcbZIP Genes

Through a combination of BLASTP and HMMER searches against the Coptis chinensis genome, we identified 55 non-redundant proteins containing the characteristic bZIP domain. These genes were systematically renamed from CcbZIP1 to CcbZIP55 based on their ascending physical locations on the chromosomes (Table 1). The encoded proteins exhibited considerable diversity in their physicochemical properties. Their lengths ranged from 81 to 893 amino acids, corresponding to molecular weights from approximately 9.56 to 93.52 kDa. The theoretical isoelectric points (pI) varied from 4.89 to 11.44, with 21 proteins (38.2%) being acidic (pI < 7) and the remainder basic. All proteins were predicted to be hydrophilic, with grand average of hydropathy (GRAVY) values between −0.44 and 0.023. Subcellular localization predictions strongly indicated that the vast majority are nuclear proteins, consistent with their putative function as transcription factors [31].

Table 1.

Characterisitic of CcbZIPS.

Gene Name Gene ID Chr Chromosome Location Gene Length (bp) ORF Length (aa) Deduced Protein Subcellular Location
Size (aa) MW (kDa) PI GRAVY
CcbZIP01 evm.model.Scaffold_63.37 6 68351006–68364660 13,654 242 81 9563.17 11.44 −0.932 Nuclear
CcbZIP02 evm.model.Scaffold_124.235 3 18006083–18007384 1301 848 283 31,878.16 6.37 −0.913 Nuclear
CcbZIP03 evm.model.Scaffold_123.165 6 93895910–93901866 5956 1252 420 47,214.42 6.69 −0.541 Nuclear
CcbZIP04 evm.model.Scaffold_65.11 3 22686753–22693929 4713 625 209 24,202.92 9.65 −0.506 Cytoplasmic
CcbZIP05 evm.model.Scaffold_56.68 6 1542536–1546055 3519 1004 335 37,011.98 5.84 −0.704 Nuclear
CcbZIP06 evm.model.Scaffold_128.187 8 88687098–88688981 1883 667 222 25,630.64 8.57 −0.919 Nuclear
CcbZIP07 evm.model.Scaffold_175.551 3 85971174–85979810 8636 941 315 35,617.36 6.64 −0.49 Nuclear
CcbZIP08 evm.model.Scaffold_161.2 2 3556701–3557183 2609 894 298 33,482.61 7.8 −0.881 Nuclear
CcbZIP09 evm.model.Scaffold_204.1 2 4085267–4090711 3335 1706 569 62,560.2 6.78 −0.895 Nuclear
CcbZIP10 evm.model.Scaffold_84.32 2 86525851–86526291 440 440 146 16,747.67 8.02 −0.784 Nuclear
CcbZIP11 evm.model.Scaffold_24.21 9 65115883–65116317 1659 473 157 17,965.1 6.29 −0.817 Nuclear
CcbZIP12 evm.model.Scaffold_165.276 2 12928906–12929914 1008 834 278 30,923.83 8.43 −0.915 Nuclear
CcbZIP13 evm.model.Scaffold_145.55 2 33619210–33623955 4745 1281 428 46,805.05 5.04 −0.711 Nuclear
CcbZIP14 evm.model.Scaffold_20.387 5 65762806–65763294 488 488 162 18,185.72 6.75 −0.466 Nuclear
CcbZIP15 evm.model.Scaffold_356.10 8 83877338–83884882 7544 2561 855 93,518.33 4.82 −0.619 Nuclear
CcbZIP16 evm.model.Scaffold_62.65 7 67370827–67376377 5550 470 157 18,044.1 9.52 −1.246 Nuclear
CcbZIP17 evm.model.Scaffold_27.71 7 85094226–85094804 578 578 192 22,328.99 6.06 −0.759 Nuclear
CcbZIP18 evm.model.Scaffold_106.121 2 10277150–10278158 1008 834 278 30,923.83 8.43 −0.915 Nuclear
CcbZIP19 evm.model.Scaffold_106.478 2 251653–259758 8105 1372 460 50,789.87 8.75 −0.603 Nuclear
CcbZIP20 evm.model.Scaffold_191.13 -- 97202–101121 3919 1214 405 44,374.16 5.51 −0.613 Nuclear
CcbZIP21 evm.model.Scaffold_252.40 3 13184165–13189401 5236 1098 368 42,110.74 6.14 −0.498 Nuclear
CcbZIP22 evm.model.Scaffold_161.83 2 2983894–2986739 2845 2059 686 74,454.55 7.18 −0.524 Endoplasmic Reticulum
CcbZIP23 evm.model.Scaffold_360.34 4 16145365–16151420 6055 1230 413 43,786.68 8.31 −0.792 Nuclear
CcbZIP24 evm.model.Scaffold_46.103 1 70302836–70303670 834 542 181 20,387.9 6.99 −0.809 Nuclear
CcbZIP25 evm.model.Scaffold_194.41 1 34592896–34597504 4608 1281 428 47,129.15 5.64 −0.636 Nuclear
CcbZIP26 evm.model.Scaffold_38.133 9 50051894–50055400 3506 1161 388 44,472.64 9.13 −0.609 Nuclear
CcbZIP27 evm.model.Scaffold_203.105 7 21177099–21183198 6099 1494 501 55,899.55 6.25 −0.588 Nuclear
CcbZIP28 evm.model.Scaffold_41.681 4 90980279–90983595 3316 864 288 31,318.5 4.78 −0.837 Nuclear
CcbZIP29 evm.model.Scaffold_370.16 -- 123764–128992 5228 1323 443 50,180.74 5.22 −0.403 Nuclear
CcbZIP30 evm.model.Scaffold_4.341 3 103306282–103317534 11,252 2178 731 81,352.76 5.28 −0.526 Nuclear
CcbZIP31 evm.model.Scaffold_78.543 8 93164141–93170657 6516 1209 406 43,075.3 5.6 −0.893 Nuclear
CcbZIP32 evm.model.Scaffold_226.12 2 2736440–2739282 2842 2056 685 74,336.37 7.18 −0.52 Cytoplasmic
CcbZIP33 evm.model.Scaffold_226.30 2 2628293–2633196 4903 914 307 33,109.99 4.89 −0.457 Chloroplast
CcbZIP34 evm.model.Scaffold_41.632 4 90521302–90524839 3537 1076 359 39,393.77 6.34 −0.772 Nuclear
CcbZIP35 evm.model.Scaffold_187.200 3 66584201–66591229 7028 732 244 27,969.38 5.31 −0.802 Nuclear
CcbZIP36 evm.model.Scaffold_282.29 4 85802034–85805554 3520 1127 376 42,433.66 6.78 −1.045 Nuclear
CcbZIP37 evm.model.Scaffold_89.303 9 57192590–57193532 942 702 231 25,845.31 5.23 −0.756 Nuclear
CcbZIP38 evm.model.Scaffold_81.149 6 96334268–96345074 10,806 1324 444 48,989.11 5.91 −0.44 Nuclear
CcbZIP39 evm.model.Scaffold_371.5 -- 73269–76031 2762 1106 369 41,307.57 6.5 −0.943 Nuclear
CcbZIP40 evm.model.Scaffold_72.208 2 9921150–9922803 1653 804 268 30,140.36 5.76 −0.432 Nuclear
CcbZIP41 evm.model.Scaffold_72.137 2 9254146–9254628 482 482 160 18,879.41 11.04 −0.831 Nuclear
CcbZIP42 evm.model.Scaffold_34.97 3 3647551–3651470 3919 1214 405 44,422.25 5.59 −0.607 Nuclear
CcbZIP43 evm.model.Scaffold_67.62 6 16899934–16900026 2600 1307 436 47,732.88 8.54 −0.918 Nuclear
CcbZIP44 evm.model.Scaffold_67.72 6 16703040–16703186 10,243 1431 480 54,036.08 8.88 −0.579 Nuclear
CcbZIP45 evm.model.Scaffold_31.662 2 4956546–4959921 3375 1706 569 62,557.24 6.78 −0.888 Nuclear
CcbZIP46 evm.model.Scaffold_11.13 8 67776316–67781705 1657 315 105 11,959.71 10.28 −0.69 Chloroplast
CcbZIP47 evm.model.Scaffold_110.91 1 75592581–75595718 3137 879 294 33,138.64 6.83 0.023 Plasma Membrane
CcbZIP48 evm.model.Scaffold_110.382 2 17308158–17319069 10,911 2674 893 97,509.61 4.79 −0.63 Nuclear
CcbZIP49 evm.model.Scaffold_1.8 -- 55368–59792 7079 1183 397 41,833.24 6.11 −0.696 Nuclear
CcbZIP50 evm.model.Scaffold_109.640 4 71801476–71806600 5124 1465 489 53,474.72 9.41 −0.791 Nuclear
CcbZIP51 evm.model.Scaffold_273.40 7 11663351–11669409 6058 1064 355 38,922.79 5.52 −0.794 Nuclear
CcbZIP52 evm.model.Scaffold_232.8 6 17932846–17933243 5953 1542 515 57,476.6 8.13 −0.797 Nuclear
CcbZIP53 evm.model.Scaffold_93.79 4 56509773–56510094 4200 1847 616 68,403.26 7.77 −0.997 Nuclear
CcbZIP54 evm.model.Scaffold_15.169 2 59307068–59307502 434 434 144 16,569.83 5.22 −0.576 Nuclear
CcbZIP55 evm.model.Scaffold_264.349 7 397066–405341 8275 1372 460 50,764.88 8.43 −0.59 Nuclear
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To elucidate the evolutionary relationships within the bZIP family, a phylogenetic tree was constructed using the full-length amino acid sequences of the 55 CcbZIPs and 50 well-characterized AtbZIPs from Arabidopsis thaliana (Figure 1). The CcbZIP proteins were classified into subfamilies based on their clustering with established A. thaliana orthologs. This analysis classified the 55 members into 12 subfamilies: A, B, C, D, E, F, G, H, I, K, M, and S. Notably, subfamilies L and O, which are present in A. thaliana, were absent in C. chinensis. Subfamilies A and I were the most expanded, containing 12 and 10 members, respectively, followed by D (9), S (7), G (4), C (2), M (2), H (2), E (2), while B, F, and K each contained a single member. Two genes, CcbZIP30 and CcbZIP33, did not cluster with any defined subfamily and were designated as orphan genes, suggesting potential lineage-specific evolution [32].

Figure 1.

Figure 1

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Phylogenetic tree of the bZIP gene family in Coptis chinensis and A. thaliana. The maximum likelihood (ML) phylogenetic tree was constructed using MEGA7.0. Subfamilies are indicated by different colored arcs. Bootstrap values from 1000 replicates are shown at key nodes. The triangle represents the CcbZIP genes.

2.2. Gene Structure and Conserved Domain Analysis

To gain deeper insights into the structural characteristics and potential functional diversification of the CcbZIP family, we systematically analyzed their conserved motifs, gene structures, and domain architectures. As shown in Figure 2, conserved motif analysis identified 10 distinct motifs (Motif 1–10) distributed across the CcbZIP proteins. The composition and sequential order of these motifs were highly conserved within members of the same phylogenetic subfamily but exhibited clear variation between different subfamilies. For instance, Subfamily A members commonly shared Motifs 1, 2, 3, and 4, whereas Subfamily I was characterized by a distinct set including Motifs 1, 5, and 6. This specific motif architecture strongly supports the reliability of our phylogenetic classification and suggests potential functional specialization among subfamilies. Analysis of the exon–intron structures further revealed substantial structural diversity (Figure 3). Genes clustered within the same subfamily generally possessed similar exon–intron organizations, reinforcing their evolutionary relationships. Notably, members of the expanded subfamilies A and I typically contained multiple introns, indicating structural complexity. In contrast, several genes from other subfamilies exhibited fewer introns or intron-less structures. This structural variation provides genomic evidence for the divergent evolution of the CcbZIP family.

Figure 2.

Figure 2

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Distribution and architecture of conserved motifs in CcbZIP proteins. The motif analysis was performed using the MEME suite, identifying 10 distinct motifs (numbered 1–10), each represented by a unique color.

Figure 3.

Figure 3

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Exon–intron structure of CcbZIP genes. Gene structures are shown aligned with the phylogenetic groups. Exons and untranslated regions (UTRs) are represented by green and yellow boxes, respectively; black lines indicate introns.

Furthermore, examination of the conserved domain architecture confirmed the presence of the characteristic bZIP domain in all 55 CcbZIP proteins (Figure 4). Beyond this core domain, numerous members harbored additional functional domains, such as regulatory or protein–protein interaction domains, which were often specific to certain subfamilies. The diversity in domain composition underscores the functional versatility within this transcription factor family and supports the notion of subfamily-specific regulatory roles acquired during evolution. Collectively, the integrated analysis of motifs, gene structures, and domains reveals a strong correlation between phylogenetic grouping and structural features, providing a comprehensive genomic foundation for understanding the functional evolution of the bZIP family in C. chinensis.

Figure 4.

Figure 4

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Conserved domain architecture of CcbZIP proteins. Protein domains were identified using the NCBI Conserved Domain Database. The bZIP domain, characteristic of this transcription factor family, is consistently present across all members and highlighted in distinct colors along with other identified functional domains.

2.3. Chromosomal Distribution, Gene Duplications and Synteny Analysis of CcbZIPs

The genomic locations of the 55 CcbZIP genes were mapped onto the nine chromosomes of C. chinensis (Figure 5). The distribution was uneven, with chromosomes 1, 2, 3, 4, 6, 7, and 8 harboring multiple genes, while chromosome 5 contained only a single gene (CcbZIP14). To understand the evolutionary mechanisms underlying the expansion of the CcbZIP family, we performed synteny analysis. Several conserved syntenic blocks were identified within the C. chinensis genome, indicative of segmental duplication events. Significant collinearity was observed between specific chromosome pairs, including Chr1-Chr4, Chr2-Chr4, Chr2-Chr7, and Chr6-Chr7 (highlighted by lines in Figure 5), suggesting these events have been crucial in the evolution of this gene family. Tandem duplication events were also detected, with relevant genes highlighted in Figure 5. These findings suggest that both segmental and tandem duplications have contributed to the expansion of the bZIP family in C. chinensis, a pattern consistent with the evolution of gene families in other plant species [33].

Figure 5.

Figure 5

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Chromosome positions and synteny analysis of CcbZIP genes. The physical positions of CcbZIP genes on the nine chromosomes of C. chinensis are shown. Genes are labeled on the right side of each chromosome. Interspecific syntenic blocks are color-coded to indicate homologous relationships between genes from different species, for visual clarity.

2.4. Expression Patterns of CcbZIP Genes Under Different Temperature and Light Conditions

To elucidate the transcriptional response of CcbZIP genes to environmental stimuli, we analyzed their expression profiles under varying temperature and light conditions using transcriptome data (Figure 6). FPKM-based heatmaps revealed that the expression of CcbZIP07, CcbZIP08, CcbZIP26, and CcbZIP27 was notably influenced by temperature, whereas CcbZIP07, CcbZIP09, CcbZIP13, CcbZIP14, CcbZIP16, CcbZIP20, CcbZIP24, CcbZIP34, CcbZIP45, and CcbZIP54 responded to different light intensities.

Figure 6.

Figure 6

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Expression profiles (log2 FPKM) of CcbZIP genes under abiotic stress. (A,B) Heatmaps showing expression of CcbZIP genes under different growth temperatures. T15: 15 °C; T25: 25 °C; T35: 35 °C; U: aerial tissues; D: subterranean tissues. (C,D) Heatmaps showing expression of CcbZIP genes under different light intensities.

We further validated these patterns using qRT-PCR under temperature stress (Figure 7). CcbZIP07 expression was up-regulated in aerial tissues (U) at 15 °C and in subterranean tissues (D) at 35 °C. CcbZIP08 showed no significant changes across temperature treatments. CcbZIP26 was up-regulated throughout the plant under low temperature (15 °C), suggesting a systemic cold response. CcbZIP27 was down-regulated in U at 35 °C but up-regulated in D under both 15 °C and 35 °C, with a more pronounced induction under cold conditions.

Figure 7.

Figure 7

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Expression patterns of selected CcbZIP genes under different temperature conditions. qRT-PCR analysis of (A) CcbZIP07, (B) CcbZIP08, (C) CcbZIP26, and (D) CcbZIP27 in aerial (left) and subterranean (right) tissues. Data are presented as mean ± SD (n = 3). Asterisks indicate significant differences compared to the control (25 °C) at the same time point (* p < 0.05; ** p < 0.005; *** p < 0.0005).

Under light stress, tissue-specific expression patterns were also evident (Figure 8). CcbZIP07 was induced in U under L0 intensity but unaffected in D. CcbZIP09 was down-regulated in all tissues under L2. CcbZIP13 was up-regulated in U under L0, while CcbZIP14 remained unchanged. CcbZIP16 decreased in U under L0 but increased under L2; in D, it increased under L0 and was stable under L2. CcbZIP20 showed no significant response. CcbZIP24 was up-regulated in U under L2 and in D under L0. CcbZIP34 was suppressed in D under L2. CcbZIP45 decreased in U under L0, and CcbZIP54 increased in U under the same condition; neither gene showed significant changes in D. Collectively, these results demonstrate that CcbZIP genes exhibit distinct and tissue-specific expression patterns under temperature and light stresses, suggesting their potential roles in mediating environmental adaptation in Coptis chinensis.

Figure 8.

Figure 8

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Expression patterns of selected CcbZIP genes under different light conditions. qRT-PCR analysis of (A) CcbZIP07, (B) CcbZIP09, (C) CcbZIP13, (D) CcbZIP14, (E) CcbZIP16, (F) CcbZIP20, (G) CcbZIP24, (H) CcbZIP34, (I) CcbZIP45, and (J) CcbZIP54 in aerial (left) and subterranean (right) tissues under varying light intensities. Data are presented as mean ± SD (n = 3). Asterisks represent significant differences compared to the control (L1) at the same time point (* p < 0.05; ** p < 0.005; *** p < 0.0005).

3. Discussion

Our genome-wide analysis successfully identified and characterized 55 bZIP transcription factors (TFs) in the non-model medicinal plant Coptis chinensis. Phylogenetic classification revealed that the CcbZIP family is organized into 12 subfamilies, exhibiting notable divergence from the model plant A. thaliana. Two key observations underscore the unique evolutionary trajectory of this gene family in C. chinensis: the absence of the L and O subfamilies, and the presence of two orphan genes, CcbZIP30 and CcbZIP33, which did not cluster with any established subfamily. The absence of entire subfamilies suggests significant lineage-specific adaptation of the bZIP functional landscape in C. chinensis. In Arabidopsis and other plants, the L and O subfamilies are often implicated in specific stress responses and developmental processes. Their loss in C. chinensis may indicate a rewiring of regulatory networks, potentially driven by selective pressures to optimize the production of specialized metabolites, such as the isoquinoline alkaloid berberine. Conversely, the marked expansion of subfamilies A and I—known to be involved in stress signaling and plant development—suggests that these functions are of critical importance in C. chinensis.

A particularly intriguing finding is the identification of CcbZIP30 and CcbZIP33. As orphan genes, they may represent rapidly evolving, species-specific TFs that have acquired novel functions [34]. Given the rich and unique alkaloid profile of C. chinensis, it is tempting to speculate that these genes could be involved in the regulation of its specialized secondary metabolism, a compelling hypothesis that warrants further functional investigation.

Supporting their potential roles in environmental adaptation, our expression analysis revealed that numerous CcbZIP genes respond dynamically to temperature and light stresses in a tissue-specific manner. For instance, CcbZIP26 was systemically up-regulated under cold stress (15 °C), while CcbZIP27 exhibited contrasting regulation between aerial and subterranean tissues, suggesting complex, tissue-specific roles in temperature response. Similarly, several genes, including CcbZIP07, CcbZIP16, and CcbZIP24, showed distinct expression patterns under different light intensities. These tissue-specific expression profiles strongly imply that CcbZIP TFs are integral components of the signaling networks that mediate environmental perception and adaptive responses in C. chinensis.

In conclusion, this study provides the first systematic characterization of the bZIP family in C. chinensis. Our results not only elucidate the evolutionary dynamics of this important gene family in a medicinal plant but also pinpoint key candidate genes—particularly the expanded A and I subfamilies and the orphan genes CcbZIP30 and CcbZIP33—for future research. Functional validation of these candidates will be crucial for understanding their specific roles in C. chinensis growth, stress adaptation, and, most importantly, the regulation of its valuable medicinal compounds.

4. Materials and Methods

4.1. Plant Materials and Stress Treatments

The Coptis chinensis cv. “Wei Lian” used in this study was employed. Two-year-old seedlings were collected from the Huangshui Town C. chinensis cultivation base in Shizhu County, Chongqing Municipality [35]. The seedlings were transplanted into soil in a growth chamber under controlled conditions: temperature of 26 °C (light)/20 °C (dark), relative humidity of approximately 60%, and a photoperiod of 16 h light/8 h darkness. After three weeks of acclimatization, when the seedlings had established and showed vigorous growth, uniformly growing seedlings were selected.

After acclimatization, uniformly growing seedlings were subjected to abiotic stress treatments for 48 h. For temperature treatments, plants were transferred to chambers set at constant temperatures of 15 °C (low temperature), 25 °C (control), or 35 °C (high temperature) during both light and dark periods, while maintaining the same light intensity and photoperiod as the acclimatization stage. For light stress treatments, plants were exposed to different light intensities (measured at the canopy level) using white LED lights: 476lx (low light, L0), 2060lx (normal light, L1), or 8340lx (high light, L2), while maintaining the temperature at 25 °C and the original photoperiod. Following treatment, aerial parts (U) and subterranean parts (D) were harvested separately, immediately frozen in liquid nitrogen, and stored at −80 °C for subsequent RNA extraction. For each treatment, three biological replicates were collected, each consisting of a pool of three individual plants.

4.2. Identification and Sequence Analysis of CcbZIP Genes

The protein sequences of 50 Arabidopsis thaliana bZIP transcription factors were used as queries to perform a BLASTP (version 2.17.0+) search (E-value ≤ 1 × 10−5) against the C. chinensis protein database (BioProject: SAMN15658057) using TBtools software (version 2.303). Simultaneously, the Hidden Markov Model (HMM) profiles for the bZIP domain (PF00170 and PF07716) were retrieved from the PFAM database and used to search the same protein database using HMMER 3.0 (E-value ≤ 1 × 10−5). All candidate sequences obtained from both searches were merged, and redundant sequences were removed. The presence of the conserved bZIP domain in each candidate was further verified using the NCBI Conserved Domain Database (CDD) and SMART database. The identified non-redundant genes were systematically renamed from CcbZIP1 to CcbZIP55 based on their ascending physical positions from chromosome 1 to 9. The physicochemical properties of the deduced CcbZIP proteins, including molecular weight and theoretical isoelectric point (pI), were predicted using the ExPASy ProtParam tool (https://web.expasy.org/protparam/, accessed on 17 December 2025). Subcellular localization was predicted using CELLO v2.5 [36].

4.3. Phylogenetic, Gene Structure, Conserved Motif Analysis, Chromosomal Distribution and Synteny Analysis

Multiple sequence alignment of the full-length amino acid sequences of the 55 identified CcbZIPs and 50 AtbZIPs was performed using ClustalW (version 2.1) with default parameters (Gap opening penalty = 10, Gap extension penalty = 0.2; and the alignment results were manually checked to correct obvious misalignments). A phylogenetic tree was constructed using the Maximum Likelihood (ML) method in MEGA7.0 software with the JTT substitution model and 1000 bootstrap replicates. The amino acid sequence of all genes was list in Table S1. The CcbZIP proteins were classified into subfamilies based on the established classification of their A. thaliana orthologs. The exon–intron structures of the CcbZIP genes were visualized based on the genome annotation file using the Gene Structure Display Server (GSDS). Conserved protein motifs were identified using the online MEME suite (version 5.5.8) with the following parameters: maximum number of motifs, 10; optimum motif width, 6 to 50 amino acids [37]. The identified motifs were annotated by searching against the NCBI-CDD database and visualized using TBtools [38]. The physical positions of the CcbZIP genes on the chromosomes were mapped using TBtools software. Gene duplication events were analyzed using the Multiple Collinearity Scan Toolkit (MCScanX, version 1.0) with default parameters [39].

4.4. RNA Extraction, Transcriptome Sequencing, and qRT-PCR Analysis

Total RNA was extracted from approximately 100 mg of ground tissue using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Genomic DNA was removed by treatment with DNase I (Takara, Kyoto, Japan). RNA integrity was verified using 1.5% agarose gel electrophoresis. For transcriptome sequencing, cDNA library construction and Illumina sequencing were performed by a commercial service (e.g., Novogene/BGI). Fastp (version 0.18.0) software was used to control the quality of raw RNA-seq data and remove the adapter [40]. The resulting clean reads were mapped to the C. chinensis reference genome, and gene expression levels were calculated as Fragments Per Kilobase of transcript per Million mapped reads (FPKM) using StringTie v1.3.1 [41,42].

For qRT-PCR validation, first-strand cDNA was synthesized from 1 μg of total RNA using the PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Japan). Gene-specific primers were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 17 December 2025) to span exon-exon junctions, and primer specificity was verified by both 1.5% agarose gel electrophoresis (single clear band) and melt curve analysis (single peak) (Supplementary Table S2 for all primer sequences). The C. chinensis actin gene was used as an internal control [35]. qRT-PCR was performed in a 20 μL reaction volume containing 10 μL of 2× qPCR MasterMix (Applied Biological Materials, Richmond, BC, Canada), 0.8 μL of each primer (10 μM), 2 μL of 1:10 diluted cDNA, and 6.4 μL of nuclease-free water. The reactions were run on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the following thermal cycling protocol: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. A melt curve analysis (65 °C to 95 °C with increments of 0.5 °C) was performed to confirm amplification specificity. All reactions were performed with three biological and three technical replicates. Relative gene expression levels were calculated using the 2−ΔΔCt method [43]. Statistical significance was determined by one-way ANOVA using GraphPad Prism version 10.0 (* p < 0.05; ** p < 0.005; *** p < 0.0005) [44].

5. Conclusions

This study presents the first genome-wide identification and analysis of the bZIP transcription factor family in the medicinal plant Coptis chinensis. We identified 55 CcbZIP genes and classified them into 12 subfamilies based on phylogenetic relationships with A. thaliana. The absence of the L and O subfamilies, common in model plants, and the discovery of two putative orphan genes (CcbZIP30 and CcbZIP33) highlight the unique evolutionary path of the bZIP family in this species. Furthermore, the distinct distribution of CcbZIP members, with significant expansions in stress-associated subfamilies A and I, suggests specific functional adaptations. Expression profiling confirmed that many CcbZIPs are responsive to temperature and light stresses in a tissue-specific manner. Collectively, these findings establish a crucial foundation for future functional studies aimed at elucidating the roles of CcbZIPs in the environmental adaptation and, potentially, the regulation of valuable benzylisoquinoline alkaloid biosynthesis in C. chinensis.

Acknowledgments

The authors would like to express their sincere gratitude to the Tsingke Biotechnology Co., Ltd., for providing technical support (primer synthesis and sequencing) and Novogene Co., Ltd., for providing transcriptome sequencing support. Thank our teachers for their academic advising and the encouragement and support of my team members.

Abbreviations

The following abbreviations are used in this manuscript:

bZIP basic leucine zipper
qRT-PCR Quantitative Real-time PCR
ABA abscisic acid
HMM Hidden Markov Model
CDD Conserved Domain Database
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Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27010431/s1.

ijms-27-00431-s001.zip (10KB, zip)

Author Contributions

Conceptualization, Y.P. and Y.W.; methodology, W.W., Z.L., L.G. and R.M.; software, W.W.; validation, W.W. and Z.L.; formal analysis, W.W.; investigation, W.W. and Z.L.; resources, Y.P. and Y.W.; data curation, W.W.; writing—original draft preparation, W.W.; writing—review and editing, Y.P., W.W. and Y.W.; visualization, W.W.; supervision, Y.P. and Y.W.; project administration, Y.P.; funding acquisition, Y.P. All authors have read and agreed to the published version of the manuscript. All the authors have read and approved the final manuscript.

Institutional Review Board Statement

Not applicable. This study did not involve human participants or animals.

Informed Consent Statement

Not applicable. This study did not involve human subjects.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This work was supported by the “Challenge System” Project for Innovation and Breakthrough in the Seed Industry of Chongqing Municipality: Coptis chinensis, the National Key Research and Development Program of China (Grant No. 2022YFD1201600) and Key Project of Chongqing Special Program for Technological Innovation and Application Development (CSTB2024TIAD-KPX0049).

Footnotes

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Associated Data

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Supplementary Materials

ijms-27-00431-s001.zip (10KB, zip)

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

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