Integrated physiological, biochemical, and transcriptomic analysis of the cold-sensitive response in Mussaenda anomala

Abstract

Mussaenda anomala exhibits high sensitivity to low-temperature stress, but its molecular adaptation mechanisms remain poorly understood. In this study, tissue-cultured seedlings were exposed to temperature gradients (25 °C as control, 12 °C, 4 °C, and 0 °C) to investigate cold-stress responses. Physiological analyses revealed increased oxidative damage (increased levels of H₂O₂, relative electrolyte conductivity (REC) and malondialdehyde (MDA)) and chloroplast/mitochondrial impairment, accompanied by downregulation of photosynthetic genes (Psb/Psa/LHCA/LHCB). The plants activated multi-level defenses, including stomatal closure, palisade tissue thickening, starch accumulation, and upregulation of starch/sucrose metabolism genes (SS/Amy). Antioxidant systems (SOD/POD/CAT) were enhanced, alongside hormonal reprogramming (ABA/JA accumulation with auxin suppression). Weighted gene co-expression network analysis (WGCNA) revealed three co-expression modules containing 12 hub genes (BKI1-like, PAO2-like, CDPK1 etc.) potentially regulating cold adaptation. These findings provide the first comprehensive molecular characterization of M. anomala’s cold stress response, offering valuable targets for breeding cold-tolerant ornamental plants.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-025-12187-4.

Introduction

Mussaenda anomala, a species of the Rubiaceae family and the genus Mussaenda, is an evergreen trailing shrub found exclusively in the Dayao Mountain of Guangxi Province and southeastern Guizhou Province in China. It is classified as a nationally protected Class I rare and endangered plant [1]. The population of this species is extremely small and narrowly distributed. Its calyx is five-lobed, with the five lobes expanded into white petal-like leaves clustered at the top of the branches. The leaf color and flower shape are exceptionally beautiful, giving it high ornamental value and broad horticultural potential [1]. As a congeneric species, Mussaenda pubescens contains medicinal compounds such as triterpenoid saponins, cycloartane saponin, and flavonoids, which have been used to develop various medications for heat-clearing, summer-heat relief, and detoxification [2, 3], However, M. anomala’s medicinal potential remains unexplored due to scarcity of population size, which has hindered its development. Observations reveal that M. anomala has a slender calyx tube, with stamens attached to the calyx tube and dense hairs at the corolla tube opening. The pistil is short and concealed within the calyx tube, severely hindering insect pollination. Notably, both artificial self-pollination and cross-pollination attempted fail to produce seeds, exacerbating the scarcity of germplasm resources and species diversity, pushing M. anomala to the brink of extinction. Current conservation efforts focuses on asexcual propagation via cuttings [4] and tissue culture [5], obtaining a large number of regenerated plants. During cultivation, it was observed that M. anomala is highly sensitive to temperature changes. Exposure to temperatures below 4 °C induces irreversible cold damage, such as leaf necrosis, shoot wilting, bark fissures, and even death of the above-ground parts (Supplementary file 1). Its poor cold resistance undoubtedly makes temperature a critical limiting factor for the growth and distribution of M. anomala. To date, no studies have investigated the physiological and molecular mechanisms underlying the response of M. anomala to low-temperature stress.

With global climate change, the irregularity, frequency, and unpredictability of extreme weather have led to frequent occurrences of chilling injury (0–15 °C) and freezing injury (< 0 °C) [6]. As a common abiotic stresses, low temperatures critically impaired plant growth, development, yield, and geographic distribution [7, 8]. To mitigate these effects, Plants adapt to low temperatures through a variety of life activities. Stomata, as channels for gas exchange between leaves and the atmosphere, have a morphology and structure closely related to stomatal conductance [9]. Plants reduce stomatal conductance, thereby inhibiting photosynthesis and transpiration [10]. Low-temperature stress disrupts photosynthesis machinery through damaging to photosystem I/II (PSI and PSII), decreasing enzyme activity, reducing electron transport rates, and destabilizing thylakoid membrane [11, 12]. Morphological responses include leaf curling, thickening ofepidermis, and densification of palisade and spongy tissue to minimize water loss under low-temperature stress [13]. Prolonged low temperatures can damage chloroplast membrane, cause leakage of stromal thylakoids [14], and lead to cellular metabolic disorders [15]. In severe cases, it can result in programmed cell death of chloroplasts and mitochondria [16]. Additionally, elevated reactive oxygen species (ROS) levels induce lipid peroxidation under low-temperature stress, reflected by malondialdehyde (MDA) accumulation [17, 18] and increased relative electrical conductivity (REC) [19], which correlate with injury severity. To counteract low-temperature damage, plants activate multi-level defense strategies, such as accumulating various osmoregulatory substances, including soluble sugars (SS), soluble proteins (SP), and free proline (Pro) [20–22], which modulate cellular osmotic potential, preserving membrane integrity [23]; Superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) eliminate excess ROS to maintain intracellular homeostasis [24, 25]; Furthermore, plants can perceive and transmit cold signals through ROS bursts and Ca²⁺ influx [26], with extracellular Ca²⁺ influx triggering rapid elevation of cytosolic Ca²⁺ concentrations [27], Ca²⁺ signals are subsequently decode by an arrray of calcium-responsive proteins [28], including calmodulin (CaM), calmodulin-like proteins (CML), calcium-dependent protein kinases (CDPK), and calcineurin B-like proteins (CBL), as well as Ca²⁺/calmodulin-regulated receptor-like kinases and Ca²⁺-dependent protein kinases (CPKs/CDPKs), which activate mitogen-activated protein kinase (MAPK) cascades to coordinate cold adaptation [16, 29]. MAPKs signaling pathway serve as central integrators of stress signaling, the MAPK signaling pathway regulates cold stress responses through the ICE1 pathway in Arabidopsis, MPK3 and MPK6 phosphorylate ICE1, thereby inhibits CBF expression, while the MEKK1-MKK1/2-MPK4 cascade enhances cold tolerance by antagonizing MPK3/MPK6 [30]. Studies have shown that the ICE-CBF-COR cascade is a CBF-dependent signaling pathway and one of the most effective mechanisms for plant cells to respond to low temperatures [31] Additionally, transcription factors (TFs), such as NAC, MYB, bZIP, and ZFP are involved in regulating COR expression and confer cold tolerance through CBF-independent signaling pathways [32]. However, phytohormones intricately modulate these cold-responsive networks [33]. ABA, as a key hormone in low-temperature responses, activates SNF1-related protein kinase 2 (SnRK2)/OPEN STOMATA 1 (OST1), which phosphorylates ICE1, inhibits HOS1-mediated ICE1 degradation, enhances ICE1 binding to the CBF promoter region, thereby upregulating COR gene expression and improving cold tolerance [34]. SA enhances cold tolerance by influencing CBF and COR expression and increasing antioxidant capacity [35]. JA promotes cold tolerance through regulating leaf senescence, the expression of CBF and downstream cold-responsive gene in maize [36, 37]. Auxin and cytokinin determine the fate of root structure and function under cold stress, thereby influencing water acquisition, nutrient transport and the stress metabolite synthesis, which affect plant cold tolerance [38]. It is evident that plants respond to cold stress through a complex transcriptional regulatory network.

Therefore, to systematically investigate cold response mechanisms in M. anomala, we used uniformly grown tissue-cultured seedlings as experimental materials, which were placed in artificial climate chambers at 25 °C, 12 °C, 4 °C, and 0 °C for 3 h, 12 h, and 24 h, respectively. Through integrating phenotypic characterization, cytological observation, physiological profiling, and transcriptomic sequencing, this study elucidated the multi-level adaptive strategies of M. anomala under chilling stress. The findings provide theoretical support for the exploration of cold-resistant germplasm resources, breeding of cold-tolerant varieties, and enhancement of cold resistance in M. anomala.

Materials and methods

Plant materials

In October 2022, healthy and disease-free stem segments of M. anomala were collected from the nursery of Qiandongnan Forestry Scientific Research Institute, Guizhou Province, China, for tissue culture. Tissue-cultured seedlings were obtained in December 2023 [5]. On March 3, 2024, uniformly grown tissue-cultured seedlings (plant height: 20 ± 1 cm; ground diameter: 5 ± 1 mm) pre-acclimatized for 15 days in greenhouse. They were acclimated for 7 days in artificial climate chambers (RGLC-P800-C3, Hefei Dascate Biotechnology Co., Ltd.) at 25 °C and 1000 lx (16/8 h light/dark cycle). The seedlings were then exposed to 12 °C (T12), 4 °C (T4), and 0 °C (T0) for 3 h, 12 h, and 24 h, respectively, with leaf samples collected at each time point. Seedlings maintained at 25 °C for 5 days served as the control group (CK); The collected leaf samples were immediately frozen in liquid nitrogen and stored at −80 °C. Each treatment included eight seedlingsand three biological replicates.

Determination of physiological and biochemical indicators

Osmotic regulators: solubal sugar (SS) content was determined using the anthrone-sulfuric acid method [39], soluble protein (SP) content was measured using the Coomassie Brilliant Blue method [40], and free proline (free Pro) content was determined using the salicylic acid method [41].

Antioxidant enzyme activities: Activities of POD, SOD and CAT were determined using the guaiacol method, nitroblue tetrazolium (NBT) method and ultraviolet absorption method, respectively [42].

Cellular damage indicators: MDA content was measured according to Wang’s method [43], H₂O₂ content was determined using Alexieva’s method [44], and REC was measured using a conductivity meter (Leici, DDS-11 A, Shanghai) [45].

Endogenous plant hormone content: Samples were sent to Nanjing Weiborui Testing Technology Co., Ltd. for determination of auxin (AUX), cytokinins (CKs), abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA) content using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

Cellular and tissue observations

Scanning electron microscopy (SEM) was performed as described by Hu et al. [46]. Samples were fixed in 2.5% glutaraldehyde solution at 4 °C for 24 h, post-fixed with 1% osmium tetroxide, dehydrated through a graded ethanol series, and dried using a critical point dryer. Samples were then mounted on sample table with conductive adhesive, coated with gold using an ion sputter coater, and imaged using a scanning electron microscope (JSM-IT700HR, Japan).

Detection of histiocytes according to the method of Chen et al. [47]. Samples were fixed in FAA solution for 24 h, dehydrated through a graded ethanol series, cleared in xylene, and sectioned at 8 μm thickness. Sections were stained with fast green and observed under a DM-3000 microscope (Leica, Germany) for imaging.

Transmission electron microscopy (TEM) was conducted according toWei et al. [48]. Samples were fixed in 2.5% glutaraldehyde solution at 4 °C for 24 h, sectioned at 60 nm thickness using an ultramicrotome (Reichert-Jung LEICA UC7, AO ULTRACUT E, USA), stained with 2% uranyl acetate followed by lead citrate, and imaged using a transmission electron microscope (FEI Tecnai G2 Spirit 120kv).

Transcriptome sequencing and data analysis

RNA extraction, quality assessment, library construction, sequencing, and bioinformatics analysis were performed at Shanghai Majorbio Biotechnology Co., Ltd. (Shanghai, China, www.majorbio.com). Total RNA was extracted from plant samples using the MJZol Total RNA Extraction Kit (Meiji, Shanghai, China). RNA quality was assessed using 5300 Bioanalyzer (Agilent), and RNA concentration was quantified using the ND-2000 (NanoDrop Technologies). Only high-quality RNA samples (OD 260/280 = 1.8–2.2, OD 260/230 ≥ 2.0, RQN ≥ 6.5, 28 S:18 S ≥ 1.0) were used for library construction. RNA-seq transcriptome libraries were prepared using 1 µg of total RNA according to the Illumina^® Stranded mRNA Prep, Ligation protocol (Illumina, San Diego, CA). Raw data were filtered using fastp (https://github.com/OpenGene/fastp). The clean reads obtained from the samples were then used for de novo assembly using Trinity (https://github.com/trinityrnaseq/trinityrnaseq/wiki). To improve assembly quality, all assembled sequences were filtered using CD-HIT (http://weizhongli-lab.org/cd-hit/) and TransRate (http://hibberdlab.com/transrate/), and assessed using BUSCO (Benchmarking Universal Single-Copy Orthologs, http://busco.ezlab.org). All transcripts obtained from the transcriptome sequencing were aligned against six major databases (NR, Swiss-Prot, Pfam, COG, GO, and KEGG) using Diamond (https://github.com/bbuchfink/diamond) with an E-value cutoff of < 1e⁻⁵ to obtain annotation information. The annotation results were statistically analyzed. The expression levels of genes and transcripts were quantified using RSEM (http://deweylab.github.io/RSEM/). To identify differentially expressed genes (DEGs) between different samples, differential expression analysis was performed using DESeq2 (http://bioconductor.org/packages/stats/bioc/DESeq2/).The resulting P-values were adjusted using the Benjamini-Hochberg method for controlling the false discovery rate (FDR). Genes with an adjusted P-value (FDR) < 0.05 and absolute log₂ fold change (|log₂FC|) > 1 were assigned as statistically significant DEGs. Co-expression network analysis was conducted using the WGCNA package in R software.

Validation by Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from samples using a kit (Qiagen, Germany). cDNA was synthesized using the StarScript Pro reverse transcription kit (www.gene-star. com), following the manufacturer’s instructions. The reaction program included pre-denaturation at 95 °C for 2 min, denaturation at 95 °C for 15 s for 40 cycles, and annealing/extension at 60 °C for 30 s. The relative gene expression levels were calculated using the 2^−△△Ct method. Using Actin as the internal reference gene, nine randomly selected DEGs were validated, and primers were designed using Primer 3.0 software (Supplementary file 2). The primer sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd. (https://www.sangon.com/). All reactions were performed with three biological and technical replicates.

Data analysis and image processing

One-way analysis of variance (ANOVA) followed by Duncan’s test to evaluate significance in SPSS Statistics 25.0 (SPSS Inc., Chicago, IL, USA), with P < 0.05 conisdered statistically significant. Figures were generated and processed using TBtools, R software, and Photoshop (2021).

Results and analysis

Effects of low-temperature stress on phenotype and cell tissue of M. anomala

The phenotype of M. anomala showed significant differences under different temperature stresses (Fig. 1). The seedlings phenotype showed no significant changes with increasing treatment time in T12, and exhibited apical bending and leaf wilting after 12 h in T4 and T0, with the chilling injury worsening after 24 h.

Fig. 1.

Phenotypic changes of M. anomala seedlings after low-temperature treatment. Bar = 10 cm. CK represents the control group grown at 25 °C, T12 indicates treatment at 12 °C, T4 at 4 °C, and T0 at 0 °C

SEM observations at the end of the treatment period revealed that the degree of stomatal closure was inversely correlated with temperature (Fig. 2). Stomata remained open in CK and T12, whereas most stomata were closed in T4 and T0. Compared to T4, a higher degree of stomatal closure and a larger proportion of completely closed stomata in T0 (Supplementary file 3), and conspicuous shrinkage of the leaf surface was also observed.

Fig. 2.

Changes in stomata of M. anomala under low-temperature treatments after 24 h. The scale bar in the first row is 50 μm, and the scale bar in the second row is 10 μm

Low-temperature stress induced significant alterations in leaf tissue cellular architecture of M. anomala (Fig. 3). The palisade and spongy tissue cells exhibited loosely arranged, irregular in shape, and characetrized by large intercellular spaces in CK. With decreasing temperature, the palisade tissue cells became more compactly, with narrower intercellular space and deeper staining.

Fig. 3.

Changes in leaf anatomical structure of M. anomala under low-temperature treatments after 24 h. Bars = 0.05 mm

Low-temperature exposure induced modifications in celluar organelles (Fig. 4).in the control (CK), cell walls and membrane structures were intact and thick, characterized by large vacuoles, abundant chloroplasts, a few starch granules and round or oval mitochondria. As the temperature decreased, cell walls thinned, vacuoles shrank, chloroplasts gradually degraded, starch granules increased in number and size, mitochondria were severely damaged, but the nucleus remained clearly visible.

Fig. 4.

Ultrastructural changes in leaf cells of M. anomala under low-temperature treatments after 24 h. The scale bar in the first row is 2 μm, and the scale bar in the second row is 1 μm. Note: CH: chloroplast; N: nucleus; M: mitochondrion, V: vacuole; S: starch grain; Lg: lipid globule; CW: cell wall

Effects of low-temperature stress on physiological indicators of M. anomala

Under low-temperature stress, seedlings exhibited significant physiological and biochemical alterations (P < 0.05), including changes in cellular damage indicators (REC, MDA, H₂O₂), antioxidant enzyme activities (SOD, POD, CAT), and osmotic regulator contents (Pro, SP, SS), as shown in Fig. 5. SS contents, CAT, and POD activities generally increased, while MDA contents showed an overall decreasing trend but remained higher than CK with decreasing temperatures and prolonged exposure. SOD activities and Pro contents initially increased and then decreased. Under T4 and T0, REC initially increased and then decreased, peaking at 12 h and reaching significantly higher than in other treatments. SP contents showed an overall increasing trend. Notably, under T12 and T4, H₂O₂ contents continued to rise with prolonged treatment time, sharply increasing after 24 h under T4. Under T0, H₂O₂ content peaked at 3 h, reaching 2.68 times that of CK, and then declined. This indicates that M. anomala responds to low-temperature stress through complex physiological and biochemical mechanisms under different temperatures.

Fig. 5.

The effect of low temperature stress on the physiological indicators of M. anomala. a REC. b MDA content. c H₂O₂ content. d SOD activity. e POD activity. f CAT activity. g SP content. h SS content. i Pro content. Different letters indicate significant differences (P < 0.05)

Endogenous hormone content showed significant differences (P < 0.05) under low-temperature stress (Fig. 6). TZR contents initially increased and then decreased, while ABA contents showed an overall increasing trend with prolonged treatment time, reaching 2.79, 1.01, and 1.63 times that of CK after 24 h. IAA contents were significantly lower than those in CK. JA and SA contents exhibited different trends under different temperatures: under T12, they initially increased and then decreased; under T4, they gradually decreased; and under T0, JA gradually increased, and SA gradually decreased and was significantly lower than CK. However, JA content in all treatments was significantly higher than CK. This indicates that phytohormone contents exhibit temperature-specific changes, involving complex interactions, crosstalk among different hormones, which collectively regulate the low-temperature adaptation mechanisms in M. anomala.

Fig. 6.

Changes in endogenous hormone contents in M. anomala under low-temperature stress. a IAA content, b ABA content, c JA content, d SA content, e TZR content

Differential gene expression analysis of M. anomala under low-temperature stress

Transcriptome sequencing results showed that a total of 89.06 Gb of Clean Data were obtained from 12 samples, with each sample yielding over 6.74 Gb of Clean Data. Each sample generated approximately 7.422 billion base pairs, with Q20 and Q30 ratios of 97.88% and 93.39%, respectively, and a GC content of 43.23% (Supplementary file 4). Principal component analysis (PCA) and correlation analysis of expression levels revealed high intra-group correlations and significant inter-group differences (Fig. 7a, b). These results indicated that the sequencing data were reliable and suitable for differential expression analysis. A total of 11,438 differentially expressed genes (DEGs) were identified. There were 5,781 DEGs in T12 vs. CK (2,273 upregulated and 3,508 downregulated); 7,182 DEGs in T4 vs. CK (3,557 upregulated and 3,625 downregulated); and 7,247 DEGs in T0 vs. CK (3,207 upregulated and 4,040 downregulated) (Fig. 7c). The number of DEGs increased as the temperature decreased. Additionally, 2,615 DEGs were common in four treatments (Fig. 7 d).

Fig. 7.

RNA-seq results, (a) Inter-sample PCA analysis, (b) Correlation analysis of expression between samples, (c) The numbers of DGEs, (d) Venn diagram of DGEs

GO enrichment analysis covered biological processes (BP), cellular components (CC), and molecular functions (MF) of DEGs. Among the top 20 enriched GO terms, DEGs were primarily annotated to the photosystem, photosystem I and II, membrane components, and transcription regulator activity (Supplementary file 5a–c).

KEGG enrichment analysis revealed that the top 20 enriched pathways included plant hormone signal transduction, photosynthesis, photosynthesis-antenna proteins, the MAPK signaling pathway in plants, and starch and sucrose metabolism (Supplementary file 5d–f). These metabolic pathways may collectively participate in the response of M. anomala to cold stress.

Effects of low-temperature stress on hormone signal transduction

Under the four treatments, 74 DEGs were enriched in the signal transduction pathways of AUX, ABA, JA, SA and CKs (Fig. 8). Among them, 35 DEGs were involved in auxin signal transduction. Low temperatures inhibited the expression of 2 AUX1 and 1 TIR1 genes; 3 AUX/IAA genes were downregulated, 1 was upregulated; 1 ARF upregulated; and 6 GH3 and 10 SAUR were downregulated.

Fig. 8.

Heat map of DEGs in hormone signal transduction pathways. The color scale represents the normalized expression levels of genes, with red indicating up-regulation and blue indicating down-regulation.Genes highlighted in green denote statistically significant DEGs (adjusted p-value < 0.05 and |log₂ (Fold Change)| ≥ 1)

In ABA signaling transduction, 2 ABA receptor proteins (PYR/PYL) and 3 PP2C genes were upregulated, 1 PYR/PYL was downregulated; 4 SnRK2 and 3 ABF5 were downregulated to varying degrees.

In JA, 4 JAZ genes were upregulated under T4, and 1 MYC2 transcription factor was activated after low-temperature treatment.

In SA, 10 DEGs responded to low-temperature stress, with 1 NPR1, 3 TGA, and 3 PR1 genes downregulated, while 1 NPR1 and 1 TGA were upregulated under T0, and 1 PR1 was upregulated under T4.

In cytokinin, 1 AHP, 2 B-ARR, and 1 A-ARR gene were downregulated. This evidence demonstrates that phytohormones mediate cold stress adaptation.

Effects of low-temperature stress on starch and sucrose metabolism

Low-temperature exposure activated starch and sucrose metabolism, a total of 40 DEGs were identified under low-temperature stress with most DEGs showing varying degrees of upregulation (Fig. 9), including β-glucosidase (β-Glu), glucosidase (Glu), sucrose synthase (SUS), starch synthase (SS), endoglucanase (EG), isoamylase (ISA), α-amylase (α-Amy), β-amylase (β-Amy), and UDP-glucose pyrophosphorylase (UGP).

Fig. 9.

Heat map of DEGs in sucrose and starch metabolism. The color scale represents normalized gene expression levels, with red indicating up-regulation and blue indicating down-regulation

Effects of low-temperature stress on the MAPK signaling pathway in plants

In the MAPK signaling pathway-plants, low temperatures activated signaling pathways mediated by H₂O₂, ROS, and hormones (Fig. 10). Under T4 and T0, the expression of key genes MPK3/6 in the MAPK signaling pathway was significantly upregulated, accompanied by coordinated high expression of three downstream ERF1 genes. Under T12 and T4, the expression of MYC2 and MKK2 was upregulated. Within the ABA-induced MAPK signaling pathway, 2 key genes MAPKKK17/18 showed upregulated expression under T4 and T0.

Fig. 10.

Heat map of DEGs in MAPK signalling pathway-plant. The color scale represents the normalized expression levels of genes, with red indicating up-regulation and blue indicating down-regulation.Genes highlighted in green denote statistically significant DEGs (adjusted p-value < 0.05 and |log₂ (Fold Change)| ≥ 1)

Effects of low-temperature stress on photosynthesis and photosynthesis-antenna proteins related DEG

In the photosynthesis pathway, 27 DEGs were identified (Fig. 11a), including 11 photosystem II (Psb), 7 photosystem I (Psa), 1 cytochrome b6-f complex (PetC), 2 photosynthetic electron transport (PetF and PetH), and 6 F-type ATP synthase (ATPF1) genes. Among these, 23 genes showed varying degrees of downregulation after low-temperature treatment. Additionally, in the photosynthesis-antenna protein pathway, 13 LHCA and LHCB genes (Fig. 11b) were all downregulated after low-temperature treatment. The widespread downregulation of genes involved in both photosynthesis and photosynthesis-antenna proteins suggests a potential suppression of photosynthetic capacity in M. anomala seedlings under low-temperature stress.

Fig. 11.

a Photosynthesis related DEGs expression heatmap, b Photosynthesis-antenna protein related DEGs expression heatmap

Construction and analysis of weighted gene co-expression networks

After filtering the 11,438 DEGs obtained from low-temperature treatment (removing genes with mean expression < 1 and coefficient of variation < 0.1), 6,767 DEGs were selected for weighted gene co-expression network analysis using the WGCNA package in R software. Based on the scale-free topology fit index (Supplementary File 6), a soft threshold power of β = 9 was selected to construct the co-expression network. Dynamic tree cutting with a cut height of 0.25 divided the network into 11 co-expression modules. Different colors represented different modules, and the gray module represented genes that could not be classified into any module (Fig. 12a). The blue, black and yellow modules were significantly correlated with multiple phenotypic traits. The blue module showed significant negative correlations with CAT (r = −0.86), H₂O₂ (r = −0.963), and SS (r = −0.818). The black module showed significant positive correlation with MDA (r = 0.926) and significant negative correlation with IAA (r = −0.951). The yellow module showed significant positive correlation with POD (r = 0.846) (Fig. 12b). These gene clusters may participate in the regulatory mechanisms of cold adaptation through oxidative stress mitigation, membrane integrity maintenance and enzymatic antioxidant defense.

Fig. 12.

Identification and correlation analysis of WGCNA module, a Hierarchical clustering tree of 11 co-expressed modules identified using WGCNA. b Correlation analysis between 11 modules and 14 physiological traits (SOD, POD, CAT, REC, MDA, H₂O₂, SP, SS, Pro, IAA, ABA, JA, SA, TZR). Note: The correlation coefficient is encoded in color from (−1) (blue) to (1) (red), and the related p-values are represented in parentheses

Further GO and KEGG enrichment analyses were conducted on the DEGs in these three modules. In the blue module, GO enrichment analysis revealed that the most significantly enriched terms were double-stranded DNA binding in molecular functions, the CCAAT-binding factor complex in cellular components, and the nucleosome assembly and DNA-directed transcription termination in biological processes (Supplementary file 7a). The black module was enriched only in molecular functions (starch binding and sequence-specific DNA binding) and biological processes (phosphorelay signal transduction system and coumarin biosynthetic process) (Supplementary file 7c). In the yellow module, the most enriched molecular function, cellular component, and biological process were DNA-binding transcription factor activity, intrinsic component of the membrane, and ethylene-activated signaling pathway, respectively (Supplementary file 7e).

KEGG enrichment results showed that the blue module was most enriched in three metabolic pathways: ribosome, plant hormone signal transduction, and glycosphingolipid biosynthesis—lacto and neolacto series (Supplementary file 7b); The stilbenoid, diarylheptanoid and gingerol biosynthesis in black module (Supplementary file 7d). The biosynthesis of various plant secondary metabolites, the MAPK signaling pathway in plants, and α-linolenic acid metabolism in yellow module (Supplementary file 7f). Additionally, plant hormone signal transduction was enriched in three modules.

Using the top 30 highly connected genes in each module as hub genes, we constructed co-expression networks for the cold stress modules of M. anomala. Each node represents a gene, and the lines between genes denoted co-expression relationships. The intramodular connectivity quantifies a gene’s topological centrality and functional significance within the module. In the blue module, five hub genes were identified: BRI1 kinase inhibitor 1-like (BKI1-like), cellulose synthase-like protein G2 (CslG2), cellulose synthase-like protein G3 (CslG3), UDP-glycosyltransferase 86A1-like (UGT86A1-like), and UDP-glycosyltransferase 83A1-like (UGT83A1-like) (Fig. 13a). In the black module, three hub genes were identified: polyamine oxidase 2-like (PAO2-like), transcription factor MYB8-like (MYB8), and GDSL esterase/lipase At2g04570-like (GDSL-like) (Fig. 13b). In the yellow module, four hub genes were identified: calcium-dependent protein kinase 2 (CDPK2), CBL-interacting serine/threonine-protein kinase 6-like (CIPK6-like), zinc finger protein ZAT10-like (ZAT10-like), and probable WRKY transcription factor 31 (WRKY31) (Fig. 13c). These genes may play crucial roles in the cold adaptation of M. anomala.

Fig. 13.

Co-expression regulatory network analysis (a) Co-expression network constructed from 30 hub genes in the blue module, (b) Co-expression network constructed from 30 hub genes in the black module, (c) Co-expression network constructed from 30 hub genes in the yellow module. Note: Circles in red was a candidate hub gene

Validation of transcriptome data by quantitative real-time PCR

To validate the reliability of the RNA sequencing results, nine DEGs were selected for qRT-PCR amplification, and their relative expression levels were measured. The results were largely consistent with the gene expression trends observed in the transcriptome sequencing data (Fig. 14 and Supplementary file 8–9), indicating that the reliability of the sequencing results are reliable in this study.

Fig. 14.

Comparison of RNA-seq and qRT-PCR expression levels. Different letters indicate significant differences (p<0.05)

Discussion

Effects of low-temperature stress on the anatomical structure of M. anomala

Chilling stress, a critical abiotic factor, profoundly impacts plant growth, developmental plasticity, and biogeographical distribution patterns. Under cold acclimation, plants initiate multilevel regulatory cascades involving epigenetic modifications, membrane lipid remodeling, and stress-responsive gene networks. Prolonged low temperatures induce metabolism dysregulation, leading to organelle ultrastructural damage and programmed cell death. Cold-induced stomatal closure hinders gas exchange, reduces chlorophyll biosynthesis and enzyme activity, alters lipid membrane states, causes chloroplast degradation, and ultimately decreases photosynthesis [49, 50]. Plant lerance to low temperatures can be enhanced by increasing palisade tissue thickness [51], promoting starch granule accumulation in chloroplasts and compact cellulose arrangement [52]. This is consistent with the results observed in this study.

Effects of low-temperature stress on physiological indicators of M. anomala

Plants deploy sophisticated physiological and biochemical strategies to combat low-temperature stress. SS accumulation maintains osmotic balance and reduces freezing point [53–55]. In this study, SS content increased as the temperature decreased, consistent with findings in cold resistance studies of Cyclocarya paliurus [56]. Additionally, significant enrichment of sucrose and starch metabolism confirmed the sugars’ dual roles as cryoprotectants and signaling molecules, activating downstream defense cascades [57, 58]. SUS, involved in sucrose catabolism, is an enzyme that cleaves sucrose to provide UDP-glucose and fructose for various metabolic pathways [59]. In this study, five SUS genes were significantly upregulated after low-temperature stress, consistent with previous findings in Arabidopsis thaliana [60]. Amy and UGP are enzymes that catalyze starch breakdown. After low-temperature stress, six Amy, two UGP and six starch synthase genes were significantly upregulated. This indicates that low-temperature stress accelerated the synthesis and degradation of starch, providing energy for plants under stress conditions. Additionally, the progressive increase in SP content over time under T0 treatment helps plants resist freezing damage. The cell membrane is a crucial protective barrier for cells. Damage from low temperatures leads to electrolyte leakage and generates free radicals, inducing lipid peroxidation of unsaturated fatty acids in the membrane, resulting in increased REC, MDA levels, and ROS bursts [61]. In this study, under severe cold stress (T4 and T0), REC increased sharply but decreased after 24 h, consistent with findings in Cucumis sativus [62]; Compared to CK, MDA content significantly increased under low-temperature stress but gradually decreased with prolonged treatment time, consistent with findings in Machilus macrocarpa [63]; Furthermore, under T12 and T4, H₂O₂ content gradually increased with prolonging treatment time, whereas under T0, it gradually decreased. These results suggested that sustained cold stress caused irreversible damage to M. anomala. In this study, POD and CAT activities gradually increased with prolonged treatment time, effectively clearing large amounts of ROS, reducing membrane permeability, and decreasing MDA production [64]. Similar results were observed in Brassica napus under low-temperature stress [65].

Effects of low-temperature stress on endogenous hormone content and hormone signal transduction p

Effects of low-temperature stress on endogenous hormone content and hormone signal transduction pathways in M. anomala

Plant growth and development under low-temperature stress are regulated by multiple plant hormones, with auxin and cytokinins playing critical roles in responding to cold stress [66, 67], and these hormones interfere with cellular activities, inhibiting meristematic division potential and reducing cell numbers [68]. This study found that IAA content was significantly lower under low temperatures compared to CK, and similar results were reported for cold stress in tomato and apple [49, 69]. In the auxin signal transduction pathway, most genes were significantly downregulated, while 6 GH3 genes were significantly upregulated. Under low-temperature stress, the regulation of MdGH3-2/12 by MdHY5 is inhibited, leading to suppressed IAA accumulation mediated by the MdHY5-MdGH3-2/12 module in apple [70]. In contrast, IAA content increased in Capsicum annuum [71] and Benincasa hispida [72], which may be related to the different regulatory mechanisms in response to low temperature in different plants. In M. anomala, cytokinins showed sensitivity to low-temperature stress, exhibiting an initial increase and then decrease, with downregulation of AHP, B-ARR and A-ARR genes. In contrast, ZT content of cold-tolerant tomatoes increased under low-temperature stress, being identified as an important hormone related to cold stress [69]. Ding et al. [73] reviewed that cytokinins negatively regulate plant cold resistance, while A-ARR, as a negative regulator in the cytokinins signaling pathway, can enhance plant freezing tolerance when overexpressed (A-ARR5/7/15) in Arabidopsis [74]. This suggests that cytokinins participated in plant low-temperature stress through a complex regulatory mechanism. ABA is a key plant hormone in responding to abiotic stress [75]. In this study, ABA content showed an overall increasing trend with prolonged low-temperature treatment. Similarly, ABA content increased in Carpobrotus edulis [76], while exogenous ABA application reduced MDA content and H₂O₂ concentration in Cynodon dactylon, enhancing its cold resistance [77]. The ABA signaling pathway is a double-negative regulatory system composed of SnRK2, PP2C, and PYR/PYL [78]. In the absence of ABA, PP2C-mediated dephosphorylation inhibits SnRK2, thereby impeding signal transduction. When ABA is abundant, ABA binding to PYR/PYL proteins inactivates PP2C, leading to SnRK2 activation. In this study, PYR/PYL and PP2C genes were significantly upregulated, which is similar to findings in alfalfa and sugarcane [79, 80], Ding et al. [34]reported that low temperatures activated SnRK2/OST1 to phosphorylate ICE1, inhibited HOS1-mediated ICE1 degradation, and enhanced ICE1 binding to the CBF promoter region, thereby upregulating COR gene expression and improving cold resistance. JA plays a crucial role in responding to abiotic stresses, including cold [37, 81, 82]. Most plant specific TIFY proteins are JA signal transduction centers, determining the transcriptional activity of JA-responsive genes and playing a crucial regulatory role in plant stress responses [83, 84]. JAZ proteins are key repressors in the JA signaling pathway, playing a significant negative regulatory role [85]. In this study, four TIFY genes in the JAZ subfamily were downregulated under T0 treatment, which may be associated with reduced inhibition of downstream MYC2-like transcription factors and subsequent activation of JA-responsive genes. Zhang et al. [86] reported that sandalwood exposed to cold stress exhibited a significant increase in endogenous JA and jasmonoyl-isoleucine levels, thereby activating CBF expression [87]. These results indicated that JA biosynthesis and signaling pathways play a positive regulatory role in cold stress tolerance. Cold stress can affect plant immune responses, a process that may involve the SA signaling pathway [88]. In the SA signal transduction pathway, NPR1 has been well-documented as one of the SA receptors that perceive and transduce SA signals [89, 90]. Normally, NPR1 is typically localized in the plant cytoplasm as a polymer, but stress conditions lead to rapid SA accumulation, causing polymeric NPR1 to be reduced to monomers and transported to the plant nucleus. In this study, SA content was significantly lower than CK after 24 h of low-temperature treatment, and 7 out of 9 SA signal transduction DEGs were suppressed by low temperatures. In contrast, SA enhanced cold tolerance in C. melo [91] and C. sativus [92], SA upregulated CBF to induce cold-tolerant TFs expression [32], and the CsNPR1-CsICE1 transcriptional regulatory cascade enhanced cold tolerance in C. sativus [92]. This indicates that SA had different responses to cold stress in different plants.

Effects of low-temperature stress on the MAPK signaling pathway

The MAPK signaling pathway serves as a central hub for integrating stress-induced intracellular signals [93], with well-documented roles in many plant cold stress responses [94–96]. Generally, the MAPK cascade consists of three sequentially acting kinases. Inactive MAPKKKs, once activated, phosphorylate MAPKKs at conserved serine/threonine residues. Activated MAPKKs further activate MAPKs by phosphorylating them [73]. In Arabidopsis, the MAPK signaling pathway regulates cold stress responses via the ICE1 pathway [30, 97]. MPK3 and MPK6 phosphorylate ICE1, thereby inhibiting CBF expression, while the MEKK1-MKK1/2-MPK4 cascade enhances cold tolerance by antagonizing MPK3/MPK6 [30]. In this study, low-temperature stress activated the ABA signaling pathway, as confirmed by changes in the expression levels of key ABA signaling regulators (PYL-PP2C-SnRK2 components). This activation further stimulated the expression of two MAPKKK18 genes. Additionally, cold-induced accumulation of H₂O₂ and ROS activated redox-sensitive transcription factors (e.g., OXI1 and NDPK), which bind to the promoter regions of MPK3. Concurrently, low temperature promoted the expression of three ethylene receptor genes (ETR), which directly enhanced MPK3 expression through ethylene-responsive elements in its promoter region. These results demonstrate that plants respond to low temperature through multiple coordinated pathways. Yu et al. [98] demonstrated that overexpression of SlMPK3 increased the antioxidant enzymes activities, elevated intracellular Pro and SS levels, and enhanced plant resistance under cold stress conditions. Jin et al. [93] also proved that plants with higher IbMPK3 expression levels exhibited stronger low-temperature tolerance. Thus, plants enhanced low-temperature tolerance by activating multiple MAPK signaling pathways under cold stress, which in turn regulated the expression of downstream defense genes.

Effects of low-temperature stress on photosynthesis and photosynthesis-antenna proteins related DEGs in M. anomala

Low temperatures compromise photosynthetic machinery through multilevel perturbations, including disrupting photosynthetic pigment complex systems,, destabilizing membrane, reducing the photochemical efficiency of PSI and PSII, decreasing enzyme activity, and dysregulating of thylakoid electron transport chain [12]. The peripheral antenna systems of PSI and PSII consist of a large number of LHC proteins, which are encoded by the LHCA and LHCB gene families [99]. As the primary cold-sensitive physiological process, photosynthetic inhibition hinders plant growth, development, and yield potential [100]. Cold-induced photosynthetic decline is correlated with stomatal closure, which restricted CO₂ diffusion and reduced transpiration rates [101, 102]. In this study, we found that low-temperature stress suppressed the expression of key genes in the photosynthetic pathway, including PSI- and PSII-related genes, F-type ATP synthase genes, and a series of photosynthesis-antenna protein genes. Similar results were reported in C. annuum [103].

Weighted gene co-expression network analysis

In this study, 12 hub genes were identified. The blue module contained 5 hub genes (BKI1-like, CslG2, CslG3, UGT86A1-like, and UGT83A1-like). BR signaling is a crucial pathway for plant cold tolerance, perceived by BR receptor kinase (BRI1) and initiating a signal transduction cascade to regulate downstream gene transcription [104, 105]. This process enhances cold tolerance through BZR1-mediated transcriptional regulation of CBF genes [106]. BKI1, as an inhibitor of BRI1, suppresses BR signaling [107]. In this study, BKI1-like expression gradually decreased with dropping temperatures, thereby alleviating the inhibition of BR signaling and enhancing the cold tolerance of M. anomala. The backbone of hemicellulose polysaccharides in plants is composed of cellulose synthase-like (Csl) proteins [108]. Csl genes and hemicellulose play significant roles in the cold tolerance [109–111]. Yuan et al. [112] reported that the MaCsl gene family in different banana varieties exhibited distinct expression patterns under low-temperature stress, with MaCslA4/12, MaCslD4, and MaCslE2 identified as candidate genes for cold tolerance in bananas. In contrast, our results indicated that CslG2 and CslG3 were downregulated after low-temperature treatment, suggesting that they negatively regulate cold stress by inhibiting hemicellulose biosynthesis. Glycosylation plays a vital role in modulating the solubility, stability, and bioactivity of various small molecules and is closely associated with plant responses to abiotic and biotic stresses [113–115]. UDP-glycosyl transferases (UGTs) catalyze the production of glycosides by transferring a carbohydrate from a nucleotide-activated monosaccharide, usually a UDP-sugar, to an alcohol, acid, thiol, or amine [116]. Studies have shown that low temperatures induced UGT91Q2 expression, mediating glucosylation involved in cold tolerance regulation in Camellia sinensis [117], Additionally, Shi et al. [118] found that overexpression of OsUGT90A1 helps protect the plasma membrane from cell damage caused by low-temperature stress. Surprisingly, in this study, two UGTs (UGT86A1-like and UGT83A1-like) were downregulated, which may negatively regulate the cold resistance of M. anomala.

Three hub genes in the black module (PAO2-like, MYB8-like, and GDSL-like) were significantly positively correlated with MDA and negatively correlated with IAA. Many abiotic stresses lead to the accumulation of polyamines (PAs), which are one of the most significant metabolic responses [119–121]. However, PAs also act as regulators of redox homeostasis and a source of ROS, generating strong oxidants such as H₂O₂ and acrolein [122], and H₂O₂ also serves as a signaling molecule involved in the stress signal transduction chain and stimulates antioxidant defense responses [123]. Polyamine oxidases (PAOs) play a crucial role in PA metabolism and maintaining cellular polyamine balance [124]. In this study, PAO2-like expression initially increased and then decreased with temperatures dropped. This may be related to the accelerated decomposition of PAs by PAOs at T12 and T4, generating large amounts of ROS to stimulate the antioxidant system to resist low-temperature stress, while its downregulation at T0 may be associated with irreversible damage caused by prolonged low temperatures to the plant. Similarly, Zhang et al. [125] reported that cold stress significantly upregulated CaPAO2 and CaPAO4 in pepper. Overexpression of these genes in Arabidopsis upregulated cold-responsive genes (AtCOR15A, AtRD29A, AtCOR47, and AtKIN1), increased antioxidant enzyme activity, and reduced the accumulation of malondialdehyde and H₂O₂, significantly enhancing freezing stress tolerance.

MYB is one of the largest members of TFs family, involved in various aspects of plant growth and development [126], and plays a positive role in low-temperature stress in multiple plants [127, 128]. In this study, MYB8-like was highly expressed under low-temperature stress. Liu et al. [129] reported that ThMYB8 overexpression in Tamarix hispida enhanced resistance by reducing lipid peroxidation and ROS accumulation in cell membranes and maintaining K/Na homeostasis. GDSL-type esterase/lipase proteins (GELPs) contained a conserved GDSL motif at the N-terminus and belonged to the lipid hydrolase superfamily. GELPs play important roles in plant growth, development, and stress responses [130]. Overexpression of SFAR4 (a novel GDSL-type esterase) [131] and CaGLIP1 [132] enhanced tolerance to osmotic and oxidative stress, respectively, while GmGELP28 overexpression [133] conferred high salt tolerance. In this study, the high expression of GELP-like under low-temperature stress may be related to enhancing the cold tolerance of M. anomala by influencing antioxidant enzyme activity and reducing MDA content to alleviate membrane permeability.

Four candidate hub genes in the yellow module (CDPK1, CIPK6-like, ZAT10, and WRKY31) were significantly positively correlated with POD. Calcium (Ca²⁺) as a universal secondary messengers in eukaryotic cells, is sensed by calmodulin (CaM), calcineurin B-like proteins (CBLs), Ca²⁺-dependent protein kinases (CDPKs), and phosphatases [16]. Among these, CDPKs sensed changes in intracellular Ca²⁺ concentrations and converted them into phosphorylation events that initiate downstream signal transduction processes [134]. OsCDPK13 and OsCPK17 are recognized as important components of Ca²⁺ signaling in low-temperature responses [135, 136], and OsCPK24 overexpression significantly increased Pro and glutathione levels during cold treatment in rice [137]. PeCPK10 from Populus euphratica [138] and VaCPK20 from Vitis amurensis [139] have also been described as positive regulators of cold stress tolerance. In this study, CDPK2 was significantly upregulated after T4 and T0 treatments, suggesting that CDPK2 may played an important role in Ca²⁺ signaling during cold stress in M. anomala. The CBL-CIPK pathway is a key player in coordinating plant adaptation and resilience, influencing plant growth and development in drought, salinity, cold, and high temperatures [140]. Yu et al. [141] reported that CBL-CIPK genes in grapevine responseed to cold stress, VaCIPK18 overexpression enhanced cold tolerance by controlling the CBF transcriptional pathway and reducing ROS production; Similarly, CuCIPK16 overexpression in citrus enhanced cold tolerance in Arabidopsis [142]. In our study, the upregulation of CIPK6-like may indicate the activation of the CBL-CIPK pathway, thereby activating the expression of downstream cold resistance genes. ZAT10 was initially identified as a cold tolerance gene belonging to the CBF-independent signaling pathway, located downstream of MAPK and upstream of ROS scavenging enzyme [143]. ZAT10 overexpression has shown to enhance tolerance to abiotic stresses in Arabidopsis, rice, wheat, and poplar [144–147]. This study found that ZAT10-like may be activated by the MAPK signaling pathway under low temperatures, regulating ROS metabolism to enhance the cold tolerance of M. anomala. WRKY-TFs are one of the most important plant-specific regulatory protein families, contributing to defense against biotic and abiotic stress responses [148, 149]. Numerous studies have confirmed that members of the WRKY TFs play an important role in plant cold stress tolerance [150, 151]. In this study, WRKY31 was significantly upregulated under cold stress. Ge et al. [152] reported that TaWRKY31 overexpression in wheat enhanced drought resistance by promoting ROS scavenging, reducing stomatal opening, and increasing the expression levels of stress-related genes. TaWRKY31 overexpression in Arabidopsis increased chlorophyll and Pro content, enhanced the activities of SOD, POD and CAT, and reduced MDA levels [153]. The results indicate that the four hub genes in the yellow module positively regulate cold stress response. Therefore, we speculate that these hub genes play an important role in the cold resistance of M. anomala.

Conclusion

In conclusion, our study fills a critical knowledge gap in the response of M. anomala to cold stress by defining its multi-layered defense strategy. The cold stress increased oxidative damage (increased levels of H₂O₂, REC and MDA) and chloroplast/mitochondrial impairment, accompanied by downregulation of photosynthetic genes (Psb/Psa/LHCA/LHCB). The plants activated multi-level defenses including stomatal closure, palisade tissue thickening, starch accumulation, and upregulation of starch/sucrose metabolism genes (SS/Amy). Antioxidant systems (SOD/POD/CAT) were enhanced alongside hormonal reprogramming (ABA/JA accumulation with auxin suppression). Furthermore, WGCNA revealed three distinct regulatory programs: (i) 5 hub genes (BKI1-like, CslG2, CslG3, UGT86A1-like, and UGT83A1-like) in blue module negatively regulated cold sensitivity; (ii) 3 hub genes (PAO2-like, MYB8-like, GDSL-like) in black module enhanced cold tolerance through antioxidant defense and membrane stabilization; and (iii) 4 hub genes (CDPK1, CIPK6-like, ZAT10, WRKY31) in yellow module positively regulated cold tolerance by upregulating calcium signaling components and stress-responsive transcription factors (Fig. 15). The identified hub genes represent high-confidence candidates for future functional validation, offering valuable genetic resources for the molecular breeding of cold-tolerant cultivars.

Fig. 15.

Regulation network model in M. anomala under low-temperature stress

Authors’ contributions

ZCP: Investigation, Validation, Writing – original draft. YLL, GYW, YY: Funding acquisition, Writing – review & editing. XT, ZWH, DYX: Investigation, Validation. SYM: Data curation, Methodology. QH: Formal analysis, Investigation. YJH: Data curation, Methodology. LLZ: Methodology, Validation. PG: Formal analysis, Resources. XW: Formal analysis, Funding acquisition. GLT: Investigation, Resources.

Funding

This manuscript was funded and supported by the National Natural Science Foundation of China–Guizhou Provincial People’s Government Karst Science Research Center Project (U1812401), the National Natural Science Foundation of China (31760124), Guizhou Province Technology Planning Project, Qiankehe Basic-ZK [2023] General 023, Guizhou Province Forest Seedling Cultivation Subsidy Project[2024-ZM-03], the New Seedling Program of Guizhou Normal University [grant code 2021-B05], and Guizhou Key Laboratory of Forest Cultivation in Plateau Mountain [Qian ke he ping tai ZSYS(2025)025].

Data availability

Raw RNA-Seq data files were deposited in SRA (https://www.ncbi.nlm.nih.gov/sra/) under the accession numbers **PRJNA1209210**.

Declarations

Competing interests

The authors declare no competing interests.