Transcriptome Analysis Uncovers Tissue-Specific Molecular Mechanisms of Cold Tolerance in Daylily (Hemerocallis fulva) Involving Hormones, Antioxidants and Soluble Carbohydrates

1. Introduction

Low temperature is one of the most destructive abiotic stresses that severely restricts the growth, biomass accumulation, flowering quality, and geographical distribution of plants [1,2]. Cold exposure impairs membrane fluidity, triggers massive burst of reactive oxygen species (ROS), disrupts photosynthetic carbon assimilation, disturbs endogenous hormone homeostasis, and blocks the biosynthesis of osmoprotective soluble carbohydrates [3,4]. Consequently, cold stress leads to leaf chlorosis, root growth inhibition, reduced flower bud differentiation, delayed anthesis, and even seedling death [5].

Phytohormones act as central signal mediators linking low-temperature perception to downstream cold-resistance gene expression, among which ethylene, gibberellin (GA) and abscisic acid (ABA) exert dominant regulatory functions [6]. Ethylene is a gaseous hormone that plays a crucial role in plant growth, development, and responses to multiple stresses, including cold [7,8]. In Arabidopsis thaliana, freezing stress tolerance is suppressed by ethylene signaling. Loss-of-function mutant ctr1-1, which constitutively activates ethylene cascades, as well as EIN3-overexpressing transgenic materials, exhibit impaired freezing resistance. Integrated genetic and biochemical analyses uncover that EIN3 mediates transcriptional repression of CBF genes [9]. Contrary to the negative regulatory role of ethylene in Arabidopsis freezing tolerance, ethylene positively boosts cold resistance in cotton [10]. Multiple genetic assays confirmed that the ethylene biosynthetic gene GhACO1 improves cold tolerance. Further molecular tests proved that GhDREB1/CBF directly transactivates GhACO1. Therefore, GhDREB1-GhACO1 module promotes ethylene accumulation to strengthen cotton cold tolerance via the CBF-dependent pathway [10]. Similarly, ethylene positively modulates cold tolerance in grapevine. Cold induces ethylene accumulation and activates the ethylene-responsive gene VaERF057. Ectopic expression of VaERF057 improves Arabidopsis cold resistance by boosting antioxidant capacity and upregulating CBF and other stress genes [11]. These existing studies have verified ethylene’s positive or negative role in cold acclimation.

Gibberellin (GA) homeostasis is balanced by synthetic and catabolic pathways, with three subfamilies of GA 2-oxidases responsible for GA degradation. In Arabidopsis thaliana, cold stress strongly induces AtGA2ox. Genetic evidence shows AtGA2ox9 mutants are freezing-sensitive, and positively regulate Arabidopsis freezing tolerance [12]. In rice, cold-induced OsWRKY53 represses anther GA biosynthesis by targeting GA synthetic gene promoters. Loss-of-function wrky53 mutants accumulate higher anther GA under cold treatment. GA counteracts SLR1-mediated inhibition of tapetum factors UDT1/TDR to sustain male fertility. Knocking out WRKY53 improves booting cold tolerance without yield penalties [13]. Generally, cold stress suppresses GA biosynthesis and activates DELLA accumulation to restrain excessive vegetative growth [14,15]. ABA is recognized as the master stress hormone governing cold tolerance [16,17]. Previous work has demonstrated that cold rapidly induces ABA accumulation in plants, for instance, the ABA synthetic gene OsNCED4 in rice is markedly upregulated under cold treatment. CRISPR/Cas9-generated osnced4 mutants exhibit cold hypersensitivity, attributed to reduced ABA levels and excessive ROS accumulation under low temperature [18]. Analogously, rice OsNAC5 confers cold resistance by transactivating OsABI5, which further orchestrates a suite of ABA- and ROS-responsive stress genes [19]. In pepper, the transcription factor CaNAC035 physically interacts with CaSnRK2.4 and directly targets the promoters of ABA biosynthetic genes CaNCED3 and CaAAO3, thereby driving ABA biosynthesis upon cold exposure [20]. Indeed, exogenous ABA enhances rice cold tolerance under cold stress and chilling acclimation. High-concentration ABA enhances seedling biomass and survival by promoting osmolyte accumulation, thereby alleviating membrane lipid peroxidation and maintaining water homeostasis. ABA also elevates SOD, POD, and CAT activities to eliminate excess H₂O₂ and O₂⁻ and strengthen ROS scavenging capacity [21].

Cold stress disrupts cellular electron transport chains and provokes excessive ROS production, which causes oxidative damage, lipid peroxidation, and protein denaturation [22]. Plants rely on two core antioxidant systems to scavenge toxic ROS: enzymatic antioxidant system and glutathione circulation system [23]. Key functional enzymes include SOD, CAT, POD, GPX, GST, and GSH. SOD converts superoxide anion into hydrogen peroxide, which is further decomposed by CAT and POD; glutathione-related enzymes eliminate lipid peroxides to alleviate secondary oxidative toxicity [6]. Under cold stress, cold-susceptible rice accumulates excessive ROS and MDA, whereas cold-tolerant rice elevates CAT and POD activities to clear ROS efficiently. OsCATC/OsPOX1 expression aligns with enzyme phenotypes, indicating CAT and POD are key ROS-scavenging factors for rice cold tolerance [24]. A cold-inducible NAC transcription factor gene DgNAC3 was cloned from chrysanthemum. Overexpressing DgNAC3 markedly enhances chrysanthemum cold tolerance. Transgenic plants accumulate less MDA, H₂O₂ and O₂⁻ under cold stress, accompanied by higher CAT, SOD, and POD activities and upregulated antioxidant genes (DgPOD, DgCAT, DgCu/ZnSOD). Therefore, DgNAC3 strengthens ROS scavenging capacity to enhance chrysanthemum cold resistance [25]. These reports indicated that the antioxidant system universally participates in cold stress mitigation.

Soluble carbohydrates function as critical compatible solutes to stabilize cell membranes, maintain cellular osmotic balance, and scavenge ROS under low temperature [26]. In Lavandula angustifolia, cold induces the expression of starch-degrading genes LaAMY, LaBAM1/3 and soluble sugar synthetic genes (LaRHM1, LaMUR4, LaUGD4), while repressing photosynthetic genes (LaPSAD1, LaPSAN, LaLHCB4.2 etc.). These genes correlate closely with starch hydrolysis, which enhances osmotic adjustment via soluble sugar accumulation [27]. During cold acclimation, dormant alfalfa sequentially accumulates starch and raffinose then re-synthesizes sucrose, accompanied by coordinated shifts in synthetase and hydrolase activities [28]. In Jatropha curcas, cold accelerates starch breakdown and soluble sugar buildup, with leaves and cotyledons showing more dramatic changes than stems and roots. Multiple sugar synthetic enzymes are activated; continuously rising invertase activity in all organs. Besides, elevated galactinol and raffinose alongside related enzyme activity confirm raffinose oligosaccharides contribute greatly to cold tolerance [29]. Cold-induced VaWRKY65 acts as a dual regulator in Vitis amurensis. It transcriptionally activates starch degradation gene VaBAM3 to accumulate soluble sugars, and directly upregulates antioxidant gene VaPOD3 to clear ROS, thereby improving cold tolerance [30]. These works lay a basis for uncovering sugar-mediated cold adaptation and signaling mechanisms.

As a core perennial ornamental crop belonging to Liliaceae, H. fulva (daylily) possesses high ornamental, ecological, and medicinal value [31]. However, low temperature in late spring and early autumn frequently restricts its vegetative propagation and reproductive development, seriously limiting its landscaping application in temperate and cold regions [32]. To adapt to cold environments, plants have evolved sophisticated multi-layered adaptive strategies, including tissue-specific transcriptional remodeling of hormone signaling, antioxidant defense, and soluble sugar metabolic pathways [33,34]. Roots and leaves execute distinct physiological functions: underground roots are the primary tissue sensing soil low temperature and responsible for water/nutrient absorption [35,36], while aerial leaves undertake photosynthesis and integrate light-temperature-hormone signals to regulate floral transition [37]. Cumulative physiological evidence has confirmed that roots and leaves deploy divergent cold acclimation strategies [38,39], yet the genome-wide tissue-specific transcriptional regulatory network of daylily under cold stress remains largely uncharacterized.

In the present study, we performed phenotypic observation and comparative RNA-seq transcriptome analysis on cold-treated and control roots and leaves of H. fulva. We systematically analyzed DEG distribution and KEGG pathway enrichment, then characterized tissue-specific transcriptional changes of ethylene, GA, ABA hormone pathways, antioxidant defense genes, and fructan/RFO/trehalose sugar metabolism genes. This work aims to reveal the tissue-specific molecular regulatory mechanisms of daylily cold acclimation, clarify the crosstalk network of cold signal, multiple phytohormones, ROS scavenging, and osmoprotective sugar metabolism, and provide theoretical basis for cold-tolerant breeding and cultivation management of daylily plants.

2. Materials & Methods

2.1. Plant Materials and Cold Stress Treatment

H. fulva uniform seedlings with consistent growth status were selected as experimental materials. Seedlings were cultivated in plastic pots filled with mixed substrate (peat soil: vermiculite = 2:1, v/v) in a greenhouse under 16 h light / 8 h dark photoperiod, 25 °C daytime / 20 °C nighttime, and 60%–70% relative humidity. Seedlings with 6 fully expanded leaves were divided into two groups: the control group (CK) and the cold stress treatment group (CD). For cold treatment, seedlings were transferred to an artificial climate incubator and subjected to continuous low temperature at 4 °C for 24 h under identical light and humidity conditions. CK seedlings were maintained at normal temperature (25 °C/20 °C day/night) without cold exposure. After treatment, fresh leaf tissues (CK-L, CD-L) and whole root systems (CK-R, CD-R) were rapidly harvested from each seedling. All samples were rinsed with sterile distilled water, wiped dry, immediately frozen in liquid nitrogen, and stored at −80 °C ultra-low temperature refrigerator for subsequent phenotypic observation and RNA-seq sequencing. Three independent biological replicates were set for each tissue group.

2.2. Phenotypic Observation and Photographing

Whole potted seedlings, complete root systems and single detached seedlings of CK and CD groups were photographed separately. Scale bars were marked uniformly in each picture: 20 cm for whole potted plants, 10 cm for root systems and single seedlings.

2.3. RNA Extraction, Library Construction and Illumina Sequencing

Total RNA was extracted from root and leaf frozen samples using the Trizol reagent kit (Invitrogen, USA) following the manufacturer’s instructions. RNA purity and integrity were detected by NanoDrop 2000 spectrophotometer and 1% agarose gel electrophoresis, respectively. RNA concentration was accurately quantified using Qubit 3.0 fluorometer. High-quality RNA samples with RIN > 6.5 were used for cDNA library construction. mRNA was enriched via oligo(dT) magnetic beads, fragmented randomly, and reverse-transcribed into first-strand cDNA using random hexamer primers. Second-strand cDNA was synthesized with DNA polymerase I, followed by end repair, A-tailing and adapter ligation. Fragments of 200–300 bp were screened by AMPure XP beads, PCR amplified, and purified to construct RNA-seq libraries. Finally, qualified libraries were sequenced on the Illumina NovaSeq 6000 platform to generate paired-end raw reads. Three biological replicates were performed.

2.4. Raw Data Quality Control and Genome Mapping

Raw sequencing reads were processed to remove low-quality reads, reads containing adapters and ambiguous N bases to obtain clean reads. Q20, Q30 and GC content of clean data were calculated to evaluate sequencing quality. The reference genome of Hemerocallis fulva was adopted as the mapping reference. Clean reads were aligned to the reference genome using HISAT2 software [40,41], and mapping statistics including total mapped reads and mapping ratio were summarized for each sample.

2.5. Differentially Expressed Genes (DEGs) Screening

Gene expression level was calculated as FPKM (Fragments Per Kilobase of transcript per Million mapped reads) based on read mapping results. Differential expression analysis between comparison groups (CK-R vs CD-R, CK-L vs CD-L) was performed using DESeq2 R package [42]. Genes satisfying |log₂(fold change)| ≥ 1 and adjusted P-value (padj) < 0.01 were defined as significantly differentially expressed genes (DEGs).

2.6. Gene Functional Annotation Analyses

Functional annotation of differentially expressed genes (DEGs) was performed against three databases: the NCBI non-redundant protein sequence database (Nr, ftp://ftp.ncbi.nih.gov/blast/db/), the Gene Ontology (GO) database (http://www.geneontology.org/), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/). KOBAS 2.0 software [43] was adopted to conduct KEGG enrichment analysis, while GO enrichment analysis for DEGs was implemented via the GOseq R package [44]. All DEGs were mapped to KEGG database orthology entries. Pathways with Q-value < 0.05 were regarded as significantly enriched pathways. Bubble plots were generated to visualize the top 20 significantly enriched pathways of root and leaf DEGs separately, with rich factor, gene number and Q-value as plotting parameters.

2.7. Data Processing and Visualization

Heat maps were drawn to display the gene expression patterns of the FPKM values by using the TBtools software [45]. Venn diagram was drawn using an online tool (https://www.bioinformatics.com.cn/static/others/jvenn/example.html) to count root-specific DEGs, leaf-specific DEGs and shared DEGs between two tissues.

3. Results

3.1. Phenotypic Responses of H. fulva to Low-Temperature Cold Exposure

To assess the morphological responses of H. fulva to cold stress, we systematically compared the aboveground and underground phenotypes between cold-treated group (CD) and non-stressed control group (CK) (Figure 1). For intact potted plants (Figure 1A), the CK seedlings exhibited a robust growth status, with abundant fully opened flowers, plump flower buds, and luxuriant green leaves. In contrast, cold-stressed CD plants displayed obvious growth retardation: fewer flower buds were formed, most floral primordia failed to bloom, and partial leaf tips presented slight withering and chlorosis compared with CK.

At the underground level (Figure 1B), the root architecture was markedly affected by low-temperature stress. The control CK possessed thicker rhizomes and a larger amount of fine fibrous roots with well-developed lateral roots. After cold exposure (CD), total root biomass was reduced, rhizome thickness decreased, and the quantity and elongation of fibrous roots were significantly suppressed. Consistent with whole-pot observations, single separated seedlings (Figure 1C) further verified cold-induced growth inhibition. The CK individuals showed expanded, stretched leaves and intact floral stalks, whereas CD seedlings presented shortened leaf length, inward-curling leaves and retarded scape development. Collectively, cold stress significantly inhibited the vegetative growth, root development and reproductive bud differentiation of H. fulva, resulting in suppressed biomass accumulation and abnormal floral development.

3.2. Transcriptome Profiles of H. fulva Plants Exposed to Cold Stress and Pathway Enrichment of DEGs

To systematically characterize the global transcriptional responses to cold stress in underground root and aboveground leaf tissues, we performed RNA-seq comparative transcriptome analysis between control and cold-treated samples, including roots (CK-R vs CD-R) and leaves (CK-L vs CD-L). On average, 27,397,800 paired-end reads were generated per sample, resulting in a total of 98 Gb of high-quality clean bases obtained from the 12 samples (Table S1). Using the H. fulva reference genome, the mapping ratio for 12 samples against the reference genome ranged from 59.75% to 82.46% (Table S2). The number of upregulated and downregulated DEGs exhibited distinct patterns between root and leaf tissues under cold treatment (Figure 2A). In root tissue (CK-R vs CD-R), a total of 9504 DEGs were detected, consisting of 4926 significantly upregulated genes and 4578 downregulated genes. In leaf tissue (CK-L vs CD-L), 6916 DEGs were identified, with 3975 genes induced and 2941 genes repressed upon cold exposure. These data indicated that cold stress triggers a broader transcriptional perturbation in roots than in leaves, and both tissues harbored more cold-activated transcripts than repressed transcripts.

A Venn diagram was constructed to dissect the overlap and tissue-specificity of cold-responsive DEGs between roots and leaves (Figure 2B). The results showed that 6339 genes were uniquely differentially expressed in cold-treated roots, while 3751 DEGs exclusively responded to cold stress in leaf tissue. 3165 genes were shared between the root and leaf comparison groups, suggesting that roots and leaves deploy largely divergent transcriptional programs to cope with low temperature, with limited conserved cold response modules across organs.

To uncover the core biological processes modulated by cold in roots, we performed KEGG enrichment bubble plot analysis for root DEGs (Figure 2C). The most significantly enriched pathways included starch and sucrose metabolism, glycolysis/gluconeogenesis, carbon metabolism, fructose and mannose metabolism. These pathways might coordinate carbohydrate catabolism to supply energy and compatible solutes for cold acclimation in roots. In addition, the pathways of plant hormone signal transduction, glutathione metabolism, alpha-linolenic acid metabolism, MAPK signaling pathway-plant were markedly enriched, which might underpin reactive oxygen species scavenging and cold stress signal transduction.

KEGG enrichment of leaf cold-responsive DEGs revealed a tissue-specific set of functional pathways distinct from those in roots (Figure 2D), including photosynthesis-associated metabolism, with carbon fixation in photosynthetic organisms being highly enriched. Plant hormone signal transduction and MAPK signaling pathway-plant, carbon metabolism, glycolysis/gluconeogenesis, fructose and mannose metabolism, and valine, leucine and isoleucine degradation remained core enriched pathways shared with roots, representing conserved cold signal transduction modules across organs. Additional stress-related pathways included alpha-linolenic acid metabolism, cysteine and methionine metabolism, and ubiquitin mediated proteolysis. These global transcriptomic differences laid a foundation for the tissue-specific regulation of hormone, reactive oxygen species scavenging, and carbon metabolism pathway genes characterized in the subsequent analyses.

3.3. Transcriptional Profiling of Ethylene Biosynthesis and Signaling Pathway in H. fulva Roots and Leaves Under Cold Stress

To characterize the transcriptional reprogramming of ethylene regulatory cascade upon cold exposure in H. fulva, transcript abundance of core genes involved in ethylene biosynthesis, signal transduction and downstream cold-responsive modules was analyzed (Figure 3). Previous investigations have established that ethylene is synthesized from S-adenosylmethionine (SAM) via two successive enzymatic reactions [46,47]. Briefly, SAM is catalyzed into 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS), followed by the oxidation of ACC to ethylene mediated by ACC oxidase (ACO) [48,49] (Figure 3A). The canonical ethylene signaling cascade has been well characterized in Arabidopsis thaliana. Core signaling constituents comprise endoplasmic reticulum-localized ethylene receptors (ETR1/2, ERS1, EIN4), the negative regulator CTR1, central membrane protein EIN2, master transcription factor EIN3, as well as downstream ERF transcription factors and functional target genes [1,50] (Figure 3A).

In the ethylene biosynthetic branch from SAM to ethylene, divergent expression patterns were observed among distinct ACS isoforms (Figure 3B). ACS1 was significantly upregulated in leaf tissues but suppressed in roots, whereas root-specific ACS7 was induced and ACS3 was markedly downregulated in leaves under cold treatment. Similarly, multiple ACO subfamily genes also show divergent expression patterns in both tissues after cold stress treatment (Figure 3C). For example, cold stress induced the expression of ACO1 in leaves, while the transcript levels of most ACO1 were reduced in roots, suggesting tissue- and isoform-specific fine-tuning of ACC and ethylene production in the daylily plant’s response to cold stress.

For ethylene signal transduction components, one ethylene receptor gene (ETR2) was transcriptionally decreased following cold stress (Figure 3D), accompanied by moderate upregulation of EIN2 in roots (Figure 3F). In addition, the ethylene signal pathway inhibitor CTR1 genes showed diverged expression profiles after cold stress, among which two genes were induced, while the other two genes were repressed in both tissues (Figure 3E). Notably, the master transcription factor gene EIN3 exhibited decreased transcript abundance (Figure 3G). Downstream targets of ethylene and cold signaling, including ERF, DREB1/2/3 and cold-regulated COR protein genes. Among them, most ERF genes were strongly upregulated under cold conditions in leaves but depressed in roots (Figure 3H). In contrast, the expression of four DREB1 and three DREB2 genes was induced in leaves, while divergent expression patterns of DREB2 genes were detected in roots (Figure 3I). The upregulation of DREB1 genes consequently facilitated the robust transcriptional activation of COR genes to initiate cold acclimation responses (Figure 3J). Collectively, cold stress might trigger isoform-specific transcription of genes involved in ethylene biosynthesis and signaling to achieve tissue-specific ethylene homeostasis, thereby improving cold tolerance of H. fulva.

3.4. Transcriptional Profiling of Gibberellin (GA) Biosynthesis and Signaling Pathway in H. fulva Roots and Leaves Under Cold Stress

GA was also involved in the cold stress response in plants [14,51]. To characterize the transcriptional reprogramming of the GA metabolic and signaling cascade in H. fulva under cold treatment, we first illustrated the full GA pathway framework, covering precursor synthesis, bioactive GA generation, GA deactivation, and downstream signal transduction (Figure 4A). The trans-geranyl-geranyl diphosphate (GGDP) is converted to ent-copalyl diphosphate (ent-CDP) by ent-copalyl diphosphate synthase (CPS), followed by successive catalysis of ent-kaurene synthase (KS), ent-Kaurene oxidase (KO), and ent-kaurenoic acid oxidase (KAO) to form GA12 [52], the universal intermediate of all GA species. Two divergent metabolic branches then synthesize bioactive GA1 (13-hydroxylation pathway) and GA4 (non-13-hydroxylation pathway) [53,54], while GA2 oxidase (GA2ox) mediated hydroxylation irreversibly converts active GAs into inactive forms (GA8, GA29, GA34, GA51) [55]. Bioactive GAs are recognized by the soluble receptor GID1; the GA-GID1 complex promotes the ubiquitination and proteasomal degradation of DELLA repressor proteins. Degradation of DELLA further releases downstream transcription factors PIF4 and DREB1 to modulate cold stress resistance [56,57].

For upstream GA biosynthetic enzymes, genes encoding KS (Figure 4B), KO (Figure 4C) and KAO (Figure 4D) exhibited distinct expression profiles between control and treatment groups. In root tissue, one KS gene was strongly upregulated under cold treatment (Figure 4B). Conversely, leaf tissue displayed the reverse pattern: constitutively high expression in CK-L was sharply suppressed in CD-L, indicating cold treatment exerts antagonistic transcriptional control over the first committed GA biosynthetic gene in underground versus aboveground organs. Three paralogs of the KO gene family were detected in roots, all induced by cold treatment (Figure 4C). Only one KO gene was expressed in leaves, and this gene was consistently induced by cold treatment in both roots and leaves, representing a universally cold-responsive upstream biosynthetic paralog. The expression levels of two KAO paralogs were significantly repressed in roots under cold treatment (Figure 4D). In leaf tissue, only Hfu02G027620 was expressed, with its expression pattern inverted relative to roots: strong induction under cold stress. This might highlight organ-specific subfunctionalization of duplicated KAO genes under stress.

Root-expressed one GA20ox gene was drastically downregulated by cold treatment (Figure 4E), suggesting weakened GA precursor synthesis in stressed roots. In leaves, three GA20ox paralogs were detected; most leaf GA20ox members accumulated higher transcripts under cold treatment compared with CK (Figure 4E), implying elevated GA biosynthesis in cold-challenged leaves. One GA2ox paralog was strongly induced in roots (Figure 4F), and in leaves, the expression of three GA2ox copies also increased after cold treatment, which would accelerate the conversion of bioactive GAs into inactive catabolites. The simultaneous activation of degrading GA enzymes in both tissues implied cold stress will exert an inhibitory effect on plant growth.

We next characterized the expression of GA receptor, DELLA repressor, and downstream transcription factor genes to evaluate cold-mediated perturbation of GA signal transduction. In roots, two GA receptor GID1C genes showed mild downregulation in roots (Figure 4G). In leaf tissue, the single detectable GID1C gene exhibited robust transcriptional induction under cold treatment. The DELLA-encoding GAI gene was moderately upregulated in root and leaf tissues (Figure 4H), indicating cold stress promoted the accumulation of the major GA signaling repressor in both roots and leaves, which would restrain GA-dependent growth responses. Two PIF4 paralogs were identified in roots: both were suppressed in roots. In leaves, one paralog was strongly and coordinately repressed by cold treatment (Figure 4I), indicating the relief of inhibitory effect on the repression of DREB1, thereby enhancing cold tolerance. Collectively, these tissue-divergent transcriptional signatures demonstrated that cold stress differentially modulates GA metabolism and signaling in underground root and aboveground leaf tissues, which likely underpins organ-specific morphological and physiological adaptation to cold exposure.

3.5. Transcriptional Profiling of Abscisic Acid (ABA) Biosynthesis and Signaling Pathway in H. fulva Roots and Leaves Under Cold Stress

ABA functions as a key stress phytohormone, and accumulating evidence reveals its essential role in plant cold tolerance through transcriptional regulation of specialized stress-responsive genes [17,58]. The biosynthesis of ABA originates from β-carotene via a series of enzymatic reactions, followed by signal transduction mediated by the PYR/PYL-PP2C-SnRK2 core module [59,60,61,62] (Figure 5A). To systematically reveal how cold stress remodels ABA pathway transcription in H. fulva, we performed transcript profiling analyses of all these genes in control and cold-treated root and leaf samples. Finally, in our data, seven ABA biosynthetic metabolic enzyme and signaling transduction genes, including β-carotene hydroxylase (CHY-β), zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), xanthoxin dehydrogenase (ABA2), molybdenum cofactor sulfurase (ABA3), ABA receptor (PLY), and protein phosphatase 2C (PP2C) were detected (Figure 5B-H).

β-carotene hydroxylase (CHY-β) catalyzes the conversion of β-carotene to zeaxanthin, representing the first committed step of ABA precursor synthesis (Figure 5B). In root and leaf tissues, two CHY-β genes were significantly upregulated under cold treatment relative to control (Figure 5B), suggesting cold stress accelerates the synthesis of zeaxanthin precursors in roots and leaves. Four ZEP genes were detected in roots. Two ZEP transcripts accumulated to higher levels under cold treatment, while the other two copies showed reduced expression after cold exposure (Figure 5C). In leaves, two ZEP enzyme genes were identified and both were repressed after cold stress (Figure 5C). NCED catalyzes the rate-limiting step of ABA biosynthesis, cleaving 9-cis-violaxanthin and 9-cis-neoxanthin to generate xanthoxin. In roots and leaves, all three detected NCED genes displayed robust transcriptional upregulation under cold stress (Figure 5D). In addition, late ABA biosynthetic genes ABA2 (Figure 5E) and ABA3 (Figure 5F) were universally cold-induced across roots and leaves. The coordinated induction of these genes might greatly promote xanthoxin generation and ABA accumulation in underground and aboveground tissues.

The core ABA signaling cascade consists of PYR/PYL/RCAR receptors, PP2C negative regulators, and SnRK2 positive kinases, which jointly mediate downstream cold-responsive gene expression. In roots, one receptor gene (PYL4) was strongly induced by cold stress (Figure 5G), which improves the capacity of root cells to perceive endogenous ABA signals. In leaves, the only PYL3 paralog showed contrasting expression change: PYL3 was moderately downregulated (Figure 5G), revealing tissue-specific differentiation of ABA signal perception. Multiple PP2C homologous genes were detected (Figure 5H). The five detected PP2C genes exhibited significant downregulation in cold-treated roots. However, all PP2C transcripts were significantly elevated in cold-treated leaves. Overall, these tissue-specific transcriptional signatures demonstrate that cold stress differentially manipulates ABA metabolism and signal transduction in underground root and aboveground leaf tissues, which mediates distinct physiological adaptation strategies for roots and leaves during low-temperature exposure.

3.6. Transcriptional Responses of Antioxidant Defense-Related Genes in H. fulva Roots and Leaves Under Cold Stress

Under cold stress, excessive reactive oxygen species, particularly O₂^⋅−, are rapidly generated, leading to lipid peroxidation and the accumulation of secondary oxidative toxins [6,63]. The coordinated action of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) constitutes the primary line of defense: SOD catalyzes the dismutation of O₂^⋅− into H₂O₂, which is subsequently detoxified to H₂O and O₂ by CAT and POD (Figure 6A). Concurrently, the glutathione system, including glutathione peroxidase (GPX), glutathione S-transferase (GST), and glutathione synthetase (GSH), mitigates cytotoxicity by scavenging lipid peroxides and secondary toxins [64]. In view of this, we investigated the transcriptomic responses of key genes involved in the antioxidant defense system under low-temperature stress. The reactive oxygen species (ROS) scavenging pathway, including enzymes such as SOD, CAT, POD, GPX, GST, and GSH, was significantly modulated by cold treatment (Figure 6).

Transcriptome analysis revealed distinct expression patterns of genes encoding these antioxidant enzymes between control (CK) and cold-treated (CD) samples (Figure 1B–G). For SOD, four genes showed upregulated expression levels in roots under cold stress (Figure 6B), while two genes exhibited contrasting expression profiles in leaves (Figure 6B). CAT-related genes were significantly upregulated in cold-treated roots, whereas leaf samples displayed downregulated expression patterns (Figure 6C). A large number of POD-encoding genes showed widespread upregulation in both root and leaf tissues under cold stress, but expressed at low levels in leaf (less than 10 fpkm), indicating a central role in H₂O₂ detoxification in roots (Figure 6D).

Among the GPX genes, their transcripts showed a significant increase in both roots and leaves after cold stress (Figure 6E). Genes involved in glutathione-mediated detoxification, including GST and GSH, showed predominantly induced expression in both tissues, suggesting enhanced capacity to neutralize lipid peroxides and oxidative toxins under cold conditions (Figure 6F, G). Collectively, these results demonstrate that cold stress triggers a comprehensive transcriptional reprogramming of the antioxidant defense system in H. fulva, with tissue-specific gene expression patterns that contribute to enhanced ROS scavenging and reduced oxidative cytotoxicity, mainly in roots.

3.7. Expression Patterns of Genes Related to Inulin Series Fructan Metabolism Pathway in H. fulva Roots and Leaves Under Cold Stress

Plants can increase their cold tolerance by accumulating the soluble sugars, such as fructans, raffinose family oligosaccharides or trehalose [65,66]. Fructans are fructose-rich polymers biosynthesized from sucrose under the catalysis of fructosyltransferases, mainly consisting of inulin-type and levan-type [67]. Inulin-type fructans are linked by β-2,1 glycosidic bonds and are widely distributed in Liliaceae and Asteraceae. By contrast, levan-type fructans are connected via β-2,6 glycosidic bonds and are predominantly present in Poaceae [68]. As a member of the Liliaceae, H. fulva preferentially accumulates inulin-type fructans [69]. Enzymes involved in the inulin series fructan biosynthesis have been identified in plant, including sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT). 1-SST catalyzes the production of 1-kestose from sucrose [70]. 1-FFT is the enzyme responsible for chain elongation of inulin series fructans [71]. Inulin series fructan degradation is catalyzed by fructan 1 exohydrolases (1-FEH) (Figure 7A).

In our data, four candidate genes encoding 1-SST were generally upregulated in both roots and leaves after cold treatment, indicating that low temperature promoted the initiation step of fructan biosynthesis (Figure 7B). Two candidate genes encoding 1-FFT were significantly upregulated in both tissues under cold stress, suggesting that low temperature enhanced fructan chain elongation (Figure 7C). Conversely, two candidate genes encoding 1-FEH were generally downregulated in roots and leaves after cold treatment, implying that low temperature inhibited fructan degradation (Figure 7D). In summary, the overall fructan metabolism in H. fulva is regulated toward the biosynthetic direction under low-temperature stress: the key biosynthetic enzyme genes (1-SST and 1-FFT) are upregulated, while the catabolic enzyme gene (1-FEH) is downregulated. This coordinated regulation ultimately promotes the accumulation of inulin-type fructans, which might contribute to cold tolerance in H. fulva.

3.8. Expression Patterns of Genes Related to Raffinose Family Oligosaccharides (RFOs) Metabolism Pathway in H. fulva Roots and Leaves Under Cold Stress

RFOs, including raffinose, stachyose, and verbascose, are biosynthesized from sucrose and activated galactosyl moieties supplied by galactinol. Increasing studies have confirmed the essential participation of galactinol and RFOs in the regulation of plant cold stress responses [26,72,73,74,75]. The galactinol-dependent pathway for RFO biosynthesis has been identified (Figure 8A). First, galactose is phosphorylated into galactose-1-phosphate under the catalysis of galactokinase (GalK). Subsequently, galactose-1-phosphate uridylyltransferase (GalT) mediates the transfer of a uridine monophosphate (UMP) moiety from UDP-glucose to galactose-1-phosphate, which generates glucose-1-phosphate and UDP-galactose. In this metabolic pathway, galactinol synthase (GolS) catalyzes the synthesis of galactinol using UDP-galactose and L-myo-inositol as substrates, representing a pivotal reaction step. Raffinose synthase (RS) then catalyzes the transfer of galactosyl residues from galactinol to sucrose to produce raffinose. Similarly, stachyose synthase (STS) mediates the addition of galactosyl groups from galactinol to raffinose for stachyose biosynthesis. Verbascose is further synthesized when verbascose synthase (VES) catalyzes the galactosyl transfer from galactinol to stachyose. In addition, RFOs can be hydrolyzed into sucrose and free galactose by alkaline α-galactosidase (AGA).

Two GalK gene transcripts were detected in our data set, showing the induced expression patterns under cold stress treatment in H. fulva roots and leaves, respectively (Figure 8B). Three and one GolS genes presented higher transcript levels in response to cold stress in H. fulva roots and leaves, respectively (Figure 8C). One and two RS genes showed elevated expression levels under cold stress treatment in H. fulva roots and leaves, respectively (Figure 8D). The expression of one STS gene was markedly up-regulated after cold stress in H. fulva roots and leaves, respectively (Figure 8E). In contrast, eight AGA transcripts were detected. In both roots and leaves, these AGA genes showed down-regulated expression levels after cold stress treatment (Figure 8F). The above data showed that under low-temperature stress, the overall RFOs metabolic pathway in H. fulva roots and leaves was regulated toward RFOs accumulation. Among them, key enzyme genes involved in the RFOs biosynthesis pathway (GalK/GolS/RS/STS) are generally upregulated in roots and leaves, promoting the biosynthesis of stress-resistant oligosaccharides such as raffinose and stachyose. In contrast, the catabolic enzyme gene AGA is universally downregulated in roots and leaves, reducing the degradation and consumption of RFOs.

3.9. Expression Patterns of Genes Related to Trehalose Metabolism Pathway in H. fulva Roots and Leaves Under Cold Stress

Trehalose is a non-reducing disaccharide formed by two glucose units linked by an α,α-1,1-glycosidic bond, and trehalose alleviates cold stress in plants by manipulating the expression of trehalose metabolism-related genes [76,77]. The trehalose pathway is conserved in plants, and is produced from uridine diphosphate (UDP)-glucose and glucose-6-phosphate by trehalose-6-phosphate synthases (TPS) and trehalose-6-phosphate phosphatases (TPP), and degraded by trehalase (TRE) (Figure 9A). In our data set, the expression levels of six TPS, five TPP, and one TRE genes were detected. Notably, four and two TPS genes were strongly induced under cold stress in H. fulva roots and leaves, respectively (Figure 9B). Two and three TPP genes showed elevated expression levels in response to cold stress in H. fulva roots and leaves, respectively (Figure 9C). The expression of one TRE gene, however, was markedly down-regulated after cold stress in H. fulva roots and leaves, respectively (Figure 9D). These results indicated that low-temperature stress significantly induced the expression of the trehalose synthase genes TPS and TPP in H. fulva, with consistent response patterns observed in both roots and leaves, thereby promoting trehalose biosynthesis. Meanwhile, cold stress suppressed the expression of the trehalose-degrading enzyme gene TRE, reducing trehalose consumption. Collectively, these changes facilitate trehalose accumulation and help H. fulva withstand low-temperature-induced damage.

4. Discussion

To survive, plants have evolved numerous mechanisms to cope with suboptimal environments; these include triggering a series of signal transduction responses and accumulating compatible metabolic substances [74,78]. Cold stress severely restricts plant growth. Plants resist cold via multiple coordinated pathways: maintaining membrane fluidity through unsaturated lipids, eliminating excess antioxidants to relieve ROS oxidative damage, adjusting phytohormones and osmoprotectant levels, and activating the ICE1-CBF-COR genetic cascade to reprogram stress-related genes [79]. For instance, cold raised MDA and H₂O₂ in Bermudagrass, while 100 μM ABA reduced these oxidative indicators, elevated chlorophyll fluorescence and PSII activity. ABA modulated ABF1 and CBF1 expressions, conferring stronger cold tolerance, especially in the cold-resistant bermudagrass genotype [80]. Under cold conditions, Arabidopsis ABA receptor PYL11 inhibited seed germination through an ABA-dependent pathway involving HAB1, OST1 and ABI5. Meanwhile, PYL11 promoted ABA-independent stomatal closure and upregulates cold-responsive genes, thereby enhancing plant freezing tolerance [81]. Meanwhile, exogenous ABA enhanced the cold resistance of winter wheat by activating antioxidant systems. ABA lowered H₂O₂ and electrolyte leakage. At −10/−20 °C, ABA elevated CAT, SOD, POD and other antioxidant enzymes in leaves and rhizomes to strengthen cold tolerance [82]. In addition, early study identified 14 soybean GmDREB1 transcription factors, most of which are strongly cold-inducible. GmDREB1 proteins activate downstream cold-responsive genes by binding DRE cis-elements. GmDREB1B;1, a key homolog of Arabidopsis DREB1A, directly upregulates soybean stress genes including GmPYL21. It enhances ABRE-dependent gene expression in an ABA-independent manner, mediating soybean cold stress tolerance [83]. In grape, ABA and cold stress synergistically activate CBF/DREB1 pathways to facilitate grape bud cold acclimation, revealing the vital role of combined environmental signals in plant cold adaptation [84]. Root priming with 0.5 mM ABA effectively alleviates cold damage of tomato seedling. ABA decreased electrolyte leakage, lipid peroxidation and H₂O₂, elevates antioxidant enzyme activity, and ion absorption under cold stress [85]. In summary, ABA serves as a central signal orchestrating multiple physiological and genetic regulatory networks to mitigate cold stress damage, and exogenous ABA can effectively boost cold resistance in various plant species.

Cold stress disrupts cellular electron transport chains and triggers massive ROS burst, which causes irreversible lipid peroxidation and cell damage. Under cold stress, cold-tolerant soybean variety had stronger antioxidant enzyme activity, higher expression of photosynthetic, trehalose synthetic and cold marker genes, less H₂O₂ and MDA, and milder leaf damage compared with cold-sensitive variety [86]. In rice, cold-induced R2R3-MYB transcription factor OsMYB4P directly activates two target genes, OsT5H and OsLEA3-2. Activated OsT5H boosts serotonin synthesis, which elevates antioxidant enzyme activity to eliminate excess ROS. OsLEA3-2 also stabilizes ROS balance for improving rice cold tolerance [87]. In wild Vitis amurensis with strong cold resistance, a cold-induced GARP transcription factor gene VaAQUILO was isolated. Overexpression of VaAQUILO enhanced cold tolerance in transgenic Arabidopsis and grape calli by elevating antioxidant enzyme activity and ROS-scavenging genes [88]. Our transcriptome data revealed that the complete enzymatic antioxidant system was activated by cold, but with clear tissue preference. Genes encoding SOD, CAT and POD displayed much stronger induction in roots than in leaves. Roots continuously absorb water and mineral nutrients under cold soil conditions, and thus face more severe oxidative stress; the robust upregulation of antioxidant enzymes provides a powerful ROS scavenging system to maintain root cell viability.

The fact that low temperature significantly induced the accumulation of fructan, RFOs, galactinol, trehalose in plant roots and leaves to enhance osmotic tolerance had also been demonstrated. Cold stress triggers massive accumulation of galactinol and RFOs in plants, galactinol synthase (GolS), the enzyme catalyzing the first step in RFO biosynthesis [26]. In Arabidopsis, AtGolS3 is specifically induced by cold stress, which is considered as a target gene of the CBF regulon. Recombinant AtGolS3 protein exhibits galactinol synthase activity in vitro, which drives the biosynthesis of galactinol and raffinose to mediate plant cold stress responses [89]. In Vitis amurensis, a cold-induced GARP transcription factor could activate cold-responsive pathways and RFO biosynthetic genes including GolS and raffinose synthase (RS), directly modulating grape VvGolS to boost osmoprotective RFO accumulation for cold acclimation [88]. RS acts as the rate-limiting enzyme for raffinose production. Raffinose-deficient zmrs mutants show weakened cold resistance and less photosynthetic carbohydrates under cold stress. Cold-induced ZmDREB1A directly binds the DRE motif of the ZmRS promoter to activate its transcription, facilitating raffinose biosynthesis and cold tolerance in maize [90]. Moreover, cold-induced nuclear activator PtrERF108 from trifoliate orange (Poncirus trifoliata) directly targets PtrRS to boost raffinose synthesis, positively regulating cold tolerance via a novel ERF108-RS regulatory module [91]. Multi-omics analyses of cold-treated beets showed cold suppresses leaf photosynthesis and induces ROS in taproots. Raffinose and its synthetic metabolites accumulate sharply in tissues, and pith raffinose level positively correlates with frost tolerance, protecting taproots against freezing injury [92]. In cucumber, CsGolS4 overexpression elevated RFOs and reduced ROS, enhancing cold tolerance; RNAi lines show lower RFOs, excess ROS, and severe cold-induced leaf wilting [93]. In petunia, a zinc finger protein, PhZFP1, positively regulated cold resistance: its overexpression strengthened freezing tolerance, while silencing increased cold sensitivity. PhZFP1 directly activated PhGolS1-1 to boost galactinol and raffinose production, and upregulates ROS-scavenging genes [94]. In grape, class II trehalose 6-phosphate synthases (TPS) lack canonical TPS catalytic activity regulating cold-resistance responses was reported. They confirmed that grape VvTPS10 positively regulates cold tolerance. VvCBF1 transcriptionally activates VvTPS10, whereas VvPUB19 mediates the ubiquitination and degradation of VvTPS10. VvTPS10 enhances trehalose levels and cold resistance through the CBF pathway, sucrose metabolism and physical interaction with VvSnRK1, uncovering a new cold-regulatory mechanism of class II TPS [95]. Overall, these osmoprotective sugars governed by GolS, RS and TPS family proteins improve plant cold resistance not only via osmotic adjustment but also by alleviating ROS oxidative stress under low-temperature environments.

Cold stress restricts sustainable plant production and disrupts plant physiological metabolism and cellular structure. In tomato, a SlRAF-like B family gene, SlRAF7, was predominantly cold-inducible, and it positively regulated tomato cold tolerance. Genetically, SlRAF7 improves cold resistance by enhancing antioxidant enzyme activities and activating the conserved ICE1-CBF-COR signaling cascade [96]. In tobacco, upon cold exposure, pre-acclimated tobacco maintained stronger photoprotection, greatly alleviating PSII photoinhibition and membrane oxidative injury [97]. OsDREB1A and OsDREB1B are cold-induced rice DREB transcription factors. OsDREB1A overexpression in Arabidopsis boosts freezing tolerance by triggering cold-responsive genes [98]. Comparative physiological and transcriptomic analyses revealed that under cold stress, cold-tolerant maize maintained intact PSII function and accumulated more secondary metabolites. Transcriptome data revealed cold-tolerant maize exhibited higher expression of genes for photosystem protection, electron transport and unsaturated fatty acid synthesis, accumulating more unsaturated fatty acids to stabilize cell membranes and achieve stronger cold tolerance [99]. Cold stress damaged bean thylakoids, disordered photosynthetic complexes, and reduced fluidity. Light recovery partly repairs bean photochemistry but cannot restore its thylakoid suprastructure [100]. Collectively, plants deploy a suite of adaptive strategies against cold stress, including activating core ICE1-CBF-COR and DREB-mediated signaling pathways, preserving photosystem integrity, enhancing antioxidant capacity, and accumulating unsaturated fatty acids to stabilize cellular membranes.

Previous transcriptome research on H. fulva leaves under low-temperature stress identified 2457 DEGs, consisting of 1253 upregulated and 1204 downregulated genes. These DEGs were annotated to participate in plant hormone signal transduction, soluble sugar biosynthesis, and pathways related to SWEET transporter genes [101]. In addition, transcriptome analysis of H. fulva roots revealed that more DEGs were identified in roots of cold-tolerant genotypes than cold-sensitive counterparts upon low-temperature treatment. These DEGs were predominantly enriched in pathways associated with secondary metabolite biosynthesis. Genes involved in Ca²⁺ signaling, MAPK cascades, transcription factors, and heat shock proteins exhibited distinct expression patterns between genotypes, which may account for their divergent responses to cold stress. Such evidence highlights the vital functions of Ca²⁺ and MAPK signaling pathways in the cold adaptation of H. fulva [102]. Furthermore, previous research verified that HfSWEET2a and HfSWEET17 from H. fulva participate in low-temperature responses. Sugars serve as the primary photosynthate and fundamental energy resource in plants. As a recently discovered class of sugar efflux transporters, SWEET proteins participate in diverse plant physiological processes, including reproductive development, senescence, and responses to cold stresses [103,104]. Nevertheless, those studies failed to characterize the specific functional genes within these pathways as well as key enzyme-coding genes in detail, which highlights the innovation of the present research.

5. Conclusions

In summary, this study systematically clarified the tissue-specific molecular regulatory network of cold acclimation in H. fulva via comparative root-leaf transcriptome analysis. Cold stress induced global transcriptional reprogramming, with distinct divergence between underground roots and aerial leaves. Roots exhibited broader transcriptional changes, stronger antioxidant activation, and conserved soluble sugar accumulation. Leaves, on the other hand, relied on the ethylene signal cascade and photosystem adjustment to resist cold damage. Three core hormone pathways (ethylene, GA, ABA) formed an organ-differentiated crosstalk module to balance growth and cold defense, while fructan, RFO and trehalose pathways coordinately synthesized osmoprotective sugars to maintain cellular stability. Our findings provide a comprehensive transcriptomic resource for cold tolerance research in daylily. The key candidate genes identified in hormone signaling, antioxidant and soluble sugar pathways can be utilized for subsequent gene function verification and molecular marker development. This work also offers theoretical guidance for the cultivation management and cold-tolerant germplasm breeding of Hemerocallis and other perennial Liliaceae ornamental flowers. Future research can focus on the functional characterization of critical cold-responsive genes and the physiological verification of hormone-sugar crosstalk under low temperature.