Genome-Wide Identification of Autophagy-Related Gene Family and Gene Expression Analysis of the CmATG8 Under Heat Stress in Chrysanthemum

Abstract

Chrysanthemum morifolium is one of the world’s four major cut flowers, valued for its ornamental and economic importance. However, high temperature stress during growth and development can reduce both yield and quality. Autophagy is a cellular self-degradation and recycling process that plays a pivotal role in maintaining homeostasis under abiotic stress. This study aimed to identify autophagy-related genes (ATGs) in C. morifolium and its close relatives, analyze their structural and evolutionary characteristics, and evaluate ATG8 expression under heat stress. Genome-wide analysis identified 130 ATGs in C. morifolium, 51 in Chrysanthemum nankingense, and 49 in Chrysanthemum lavandulifolium. Genes within the same subfamily exhibited conserved structures and domains, with fragment duplication contributing to ATG expansion. Expression profiling showed that ATG8 genes were the most highly expressed and displayed tissue specificity, while heat stress induced their transcription, peaking at 48 h. These findings provide a comprehensive genomic resource for Chrysanthemum ATGs and indicate a potential role for ATG8 in heat stress responses, offering a basis for future studies aimed at improving thermotolerance in this ornamental crop.

1. Introduction

In plants, autophagy occurs in three main forms [1]. Macroautophagy forms double-membrane autophagosomes around cellular components, which fuse with vacuoles or lysosomes for degradation and recycling [2]. Microautophagy involves direct invagination of the vacuolar or lysosomal membrane to engulf cytoplasmic components without forming double-membrane autophagosomes [3]. Mega autophagy occurs under programmed cell death or severe stress, where vacuolar rupture releases hydrolytic enzymes to rapidly degrade cellular contents [4]. In plants, autophagy is maintained at low basal levels under normal conditions but is rapidly activated by abiotic stresses such as nutrient deprivation, drought, salinity, and heat [5,6,7,8,9].

Accumulating evidence has demonstrated that the expression of ATGs plays a critical role in modulating plant adaptation to various environmental stresses. For instance, in Arabidopsis thaliana, mutants deficient in ATG5, ATG7, or NBR1 exhibit compromised cellular membrane integrity and increased damage to photosystem II (PSII) under heat stress compared to wild-type plants. These mutants also accumulate higher levels of insoluble protein aggregates and display significantly lower survival rates under drought, salinity, and oxidative stress conditions [10]. In contrast, plants overexpressing AtATG5 or AtATG7 demonstrate enhanced autophagic activity, delayed senescence, prolonged flowering periods and lifespan, and increased resistance to the necrotrophic pathogen Alternaria brassicicola, as evidenced by suppressed fungal growth in transgenic lines [11]. Moreover, transgenic Arabidopsis, tobacco, and grapevine calli overexpressing VvATG6, generated via Agrobacterium-mediated transformation, exhibit significantly enhanced antioxidant enzyme activities following copper (Cu) exposure. These lines also show reduced accumulation of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), along with downregulated expression of ROS-generating genes RbohB and RbohC, thereby improving Cu stress tolerance [12]. In Solanum lycopersicum, the heat shock transcription factor HsfA1 plays a key role in drought tolerance. Research has shown that SlHsfA1a binds to the promoter regions of ATG10 and ATG18f, suggesting that it enhances autophagy by regulating ATG gene expression, thereby increasing the plant’s ability to withstand drought stress [13].

Under abiotic stress conditions, plants often experience an accumulation of misfolded or unfolded proteins within the endoplasmic reticulum (ER), thereby triggering the ER stress response [14]. Autophagy, a major intracellular degradation pathway for macromolecules, has been shown to be activated by ER stress through the ER stress sensor INOSITOL-REQUIRING ENZYME 1b (IRE1b), which facilitates the sequestration and transport of ER fragments to the vacuole for degradation. Notably, heat stress can induce ER stress, thereby promoting autophagy activation [15]. ATG8 is essential for autophagosome formation, selective autophagy execution, and cargo recognition. Through interactions with specific autophagy receptors, ATG8 mediates the targeted degradation of damaged organelles and protein aggregates [16]. Studies on the A. thaliana ATG8 gene family have shown its involvement in plant growth, development, and stress adaptation [17,18,19]. ATG8 also contributes to the recovery of the Golgi apparatus following heat stress [20,21] and directly interacts with the selective autophagy receptor NBR1. In A. thaliana, mutants lacking NBR1 accumulate high levels of insoluble proteins under heat stress, highlighting its role in maintaining protein homeostasis [22]. In Capsicum annuum, silencing the CaATG8c gene reduces chlorophyll levels and increases malondialdehyde content under heat stress, ultimately lowering the plant’s heat tolerance [23]. In contrast, overexpressing SlATG8f in S. lycopersicum weakens the plant’s overall heat tolerance but significantly improves pollen viability and reduces damage to chloroplasts and mitochondria under high temperatures [24]. These findings suggest that ATG8 is not only a central regulator of autophagy but also plays a vital role in helping plants cope with heat stress.

With its rich colors, diverse flower forms, and extended blooming period, Chrysanthemum is widely used in landscape design, potted displays, and the cut flower market. However, under the influence of global climate change, high temperature stress has become one of the major limiting factors affecting its growth, development, and industrial production [25]. In Chrysanthemum cultivation, the optimal growth temperature is around 25 °C; when ambient temperatures exceed this threshold, plant growth and development slow markedly, and at 40 °C, growth nearly ceases. During the vegetative growth stage, chrysanthemums frequently encounter high summer temperatures, resulting in growth arrest, delayed flowering, and reduced flower coloration, all of which are key factors in limiting flowering quality and yield [26]. Therefore, enhancing thermotolerance in Chrysanthemum has become an urgent priority for ensuring the sustainability of the industry. Autophagy is a critical cellular process in plant stress responses; however, the ATG gene family remains largely uncharacterized in Chrysanthemum and its relatives. This study presents a comprehensive bioinformatic and tissue-specific expression analysis of ATG genes, with a focus on their transcriptional responses to heat stress. The results provide essential genetic resources and a basis for the molecular breeding of a heat-tolerant chrysanthemum via genetic engineering.

2. Results

2.1. Gene Family Identification and Physicochemical Property Analysis

A total of 130, 49, and 51 ATG gene family members were identified in C. morifolium, C. nankingense, and C. lavandulifolium, respectively. Within each subfamily, genes were named sequentially according to their sequence ID order. Based on protein domain features, the CmATG gene family is classified into 22 subfamilies (Supplementary Table S1). The length of the proteins encoded by the CmATG family ranges from 86 (CmATG10e) to 2464 amino acids (CmTORd), with molecular weights spanning from 9847.33 Da (CmATG10e) to 276,398.94 Da (CmTORd). The predicted results show that the isoelectric points of CmATG proteins range from 4.58 (CmATG3a) to 9.63 (CmATG12c), the instability index ranges from 30.58 (CmATG8b) to 76.48 (CmATG13a), and the aliphatic index varies between 65.33 (CmATG13a) and 106.59 (CmATG7d). The grand average of hydrophilicity is negative for all but nine CmATGs, suggesting that the majority of CmATG proteins are hydrophilic.

The CnATG and ClATG gene families contain 21 subfamilies each (Supplementary Tables S2 and S3). The proteins encoded by the CnATG and ClATG family range in length from 96 (CnATG12) to 2507 amino acids (CnTORa), and from 96 (ClATG12) to 2464 amino acids (ClTORb), with molecular weights ranging from 10,664.2 Da (CnATG12) to 281,609.62 Da (CnTORa), and from 10,567.09 Da (ClATG12) to 276,458.09 Da (ClTORb). The isoelectric points, instability indices, and aliphatic indices of the CnATG and ClATG proteins are similar to those of the CmATG proteins, with the grand average of hydrophilicity values all being negative.

2.2. Phylogenetic Analysis

To explore the evolutionary relationships among ATG genes, a phylogenetic tree was constructed in MEGA 11.0 based on 130 CmATG protein sequences, together with 45 AtATG proteins and the identified 49 and 51 ATG proteins from C. nankingense and C. lavandulifolium, respectively. The phylogenetic analysis (Figure 1) classified the ATG proteins into six major groups. Most homologous genes clustered together within the same subgroup, except for members of the ATG7, ATG14, and ATG20 subfamilies, which were distributed across different branches. The evolutionary relationships among species varied between subfamilies; however, compared to A. thaliana, C. morifolium ATG proteins exhibited a closer evolutionary relationship with those of C. nankingense and C. lavandulifolium.

2.3. Gene Structure, Conserved Motif, and Domain Analysis of the ATG Gene Family

The CmATG genes were classified based on their associated protein kinase complexes, and their gene structures, conserved domains, and motifs were analyzed and visualized. The results (Figure 2) show that genes within the same subfamily exhibit specific characteristics in terms of gene structure, conserved domains, and motif composition. The number of introns in the CmATG gene family ranged from 0 to 55, while the number of exons varied from 1 to 54. For example, CmATG6b has no introns and only one exon, whereas CmTORb contains 55 introns and 54 exons. In the CnATG genes, intron numbers ranged from 2 (CnATG10b) to 58 (CnTORa), and in the ClATG genes, intron numbers ranged from 0 (ClATG6) to 56 (ClTORa and ClTORb) (Supplementary Figures S1 and S2).

Motif analysis of the ATG proteins using MEME revealed that motifs varied across CmATG subfamilies, with similar motif patterns and quantities within the same subfamily (Figure 2). When analyzing all CnATG and ClATG subfamilies together, we identified 25 distinct motifs, though some members were missing certain motifs (Supplementary Figures S3 and S4). This suggests that the structural differences between ATG proteins across subfamilies make it more meaningful to analyze motifs according to their associated protein kinase complexes.

In terms of conserved domains, all CmATG proteins within the same subfamily shared the same conserved domains, except for CmATG18 (Figure 2). In the CmATG18 subfamily, six members contained both BCAS3 and WD40 domains, while 17 members had only the WD40 domain. CmATG18h had only the BCAS3 domain. Similar patterns were observed in the ClATG18 and CnATG18 subfamilies (Supplementary Figures S3 and S4), suggesting functional diversity among these members.

2.4. Chromosomal Localization and Synteny Analysis of ATG Gene Family Members

Chromosomal localization analysis of Chrysanthemum showed that CmATG10d and CmATG12b have not yet been assigned to chromosomes. The remaining CmATG genes are distributed unevenly across the 27 chromosomes of Chrysanthemum. Chromosomes Chr13 and Chr15 each contain up to 10 CmATG genes, while chromosomes Chr19, Chr20, and Chr21 have the fewest, with only one CmATG gene each (Figure 3). The CnATG gene family has six members (CnATG7d, CnATG7f, CnATG8f, CnATG10a, CnVTI12a, CnVTI12c) that have not yet been assigned to chromosomes. Additionally, chromosome Chr3A in C. nankingense and LG03 in C. lavandulifolium lacked any ATG gene family members (Supplementary Figures S5 and S6).

An intra-species synteny analysis of the CmATG gene family was performed, followed by an inter-species synteny analysis comparing C. nankingense and C. lavandulifolium. The results of the intra-species synteny analysis showed that the CmATG gene family does not contain tandem repeats but rather expands through segmental duplications, forming 252 pairs of syntenic genes (Figure 4a). The inter-species synteny analysis revealed 149 and 196 pairs of syntenic genes between CmATG and CnATG, and between CmATG and ClATG. Typically, three CmATG genes correspond to one CnATG and ClATG (Figure 4b).

2.5. Analysis of Cis-Regulatory Elements in the Promoters of the Chrysanthemum ATG Gene Family

Cis-regulatory element analysis revealed 18 common cis-regulatory elements in the Chrysanthemum ATG gene family, which were classified into five categories (Figure 5, Supplementary Figures S7 and S8). These include elements associated with abiotic stress (five elements), plant hormone responses (seven elements), plant growth and development (two elements), light response, and transcription factor binding sites (three elements). The CmATG genes contain diverse cis-regulatory elements linked to abiotic stress responses, including ARE (anaerobic response element), MBS (MYB-binding site involved in drought response), and LTR (low-temperature responsiveness), as well as hormone-responsive elements such as ABRE (ABA-responsive element), TGA (auxin-responsive element), and CGTCA motifs (MeJA-responsive element). These elements suggest a significant role for CmATG genes in mediating stress adaptation and hormonal regulation in chrysanthemum. Furthermore, 29 members of the CmATG gene family harbor growth- and development-related elements, such as CAT-box (meristem expression) and O2-site (zein metabolism regulation), indicating their potential involvement in developmental processes. Notably, 105 CmATG genes possess binding sites for key transcription factors, including MYB, MYBHv1, and ATBP-1, underscoring their regulatory complexity and potential involvement in transcriptional control under various physiological and stress conditions. These results suggest that CmATG genes may play a pivotal role in coordinating chrysanthemum responses to abiotic stresses and hormone-mediated regulatory networks, contributing to its stress resilience and developmental regulation.

2.6. Tissue-Specific Expression of the Chrysanthemum ATG Gene Family

To confirm whether the identified CmATG genes are normally expressed in Chrysanthemum, the expression of 130 ATG gene members in different tissues was analyzed. Expression data for the ATG genes were obtained from the Chrysanthemum transcriptome database [27]. The transcription levels of all members were compiled (Supplementary Table S4). Among these, 111 genes showed differential expression across different tissues, while the remaining 19 genes had FPKM values close to 0 or 0 in all tissues. FPKM (fragments per kilobase of transcript per million mapped reads) was used to quantify transcript levels; this metric normalizes read counts for both transcript length and sequencing depth, and values near zero indicate negligible or no detectable expression.

The expression levels of all detected genes were analyzed and visualized (Figure 6a). The results revealed significant variation in the expression levels of different CmATG family members. Genes from the CmATG8 and CmVTI12 subfamilies generally exhibited higher expression levels, while genes from other subfamilies, such as CmATG1a, CmATG1b, and CmATG9c, showed lower expression levels. Analysis of the expression in various tissues showed that CmATG8b was prominently expressed in tubular flower petals, ray florets, pistils of ray florets, and shoot apical meristems. In tubular flower pistils, CmATG8n had the highest expression, while CmATG8d was most highly expressed in tubular flower stamens, roots, apical tissues, and stems. In leaves, CmATG8i was the most highly expressed gene.

A differential expression analysis of the 18 CmATG8 family genes across various tissues revealed that CmATG8s showed a trend of increasing expression followed by a decrease during different stages of flower bud development. The highest transcription levels were observed in 4 mm flower buds, suggesting that autophagy activity is higher during the early stages of flower bud development under normal growth conditions (Figure 6b). This implies that the CmATG8 subfamily plays a role in the differentiation and formation of flower buds.

2.7. Expression Analysis of CmATG8 Subfamily in Response to Heat Stress

The CmATG8 gene family exhibited relatively high transcript levels across various tissues, suggesting its potential involvement in stress-related processes. To investigate their roles in heat stress response, the expression dynamics of CmATG8 genes were examined by qRT-PCR in chrysanthemum leaves sampled at eight time points within a 72 h period following heat treatment. As shown in Figure 7, all CmATG8 members responded to heat stress, though with distinct temporal expression patterns that could be grouped into four major categories.

CmATG8a, CmATG8b, CmATG8c, CmATG8d, CmATG8h, CmATG8k, CmATG8l, CmATG8n, and CmATG8p displayed low expression during the initial 24 h of treatment, followed by a pronounced upregulation at 48 h and a subsequent decline by 72 h. Among these, CmATG8c and CmATG8d showed the most substantial increases, with transcript levels rising by 13.4-fold and 11.2-fold, respectively, while CmATG8k and CmATG8l increased by 8.9-fold and 8.1-fold, respectively. CmATG8f, CmATG8g, CmATG8i, CmATG8j, and CmATG8q exhibited a rapid early response, with peak expression observed at 1 h or 3 h post-treatment. Notably, CmATG8g expression increased by 3.1-fold at 1 h. CmATG8e and CmATG8m showed a distinct pattern of response; their expression levels began to increase at 12 h, initially declining from 12 to 24 h before rising again from 24 to 48 h. Most CmATG8 genes reached their lowest expression levels at 24 h of treatment. Interestingly, CmATG8o exhibited a distinct and sustained upregulation: its transcript levels rose approximately 4.5-fold at 1 h and 3 h, peaked at a 7.5-fold increase by 12 h, declined slightly at 24 h, and then continued to rise between 48 h and 72 h, ultimately reaching a 15.4-fold increase relative to the untreated control. These findings suggest that CmATG8o may play a critical role in mediating Chrysanthemum’s response to heat stress.

3. Discussion

Autophagy is an essential process in plant growth and development, playing a vital role in maintaining cellular homeostasis, delaying senescence, and enhancing stress tolerance. This complex biological mechanism is orchestrated by a series of ATG proteins with distinct functions. To date, the ATG gene family has been identified and analyzed in multiple plant species, including Arabidopsis thaliana (39 genes) [28], Oryza sativa (33 genes) [29], Camellia sinensis (80 genes) [30], Triticum aestivum (108 genes) [31], Medicago truncatula (39 genes) [32], Nicotiana tabacum (30 genes) [33], Fagopyrum tataricum (49 genes) [34], Beta vulgaris (29 genes) [35], Lycium ruthenicum (36 genes) [36], and Solanum tuberosum (29 genes) [37].

Based on whole-genome data, 130 ATG genes were identified in C. morifolium, 49 in C. nankingense, and 51 in C. lavandulifolium. The observed gene numbers aligned with the species’ ploidy levels, indicating a possible correlation between gene family expansion and genome duplication. Gene copy number analysis showed that the expansion of the CmATG8 subfamily is independent of ploidy level. C. morifolium contains 17 ATG8 genes, while C. nankingense and C. lavandulifolium contain eight and nine copies. ATG8 is crucial for autophagosome membrane formation via covalent binding to phosphatidylethanolamine (PE), and its essential role under environmental stresses likely subjects it to strong purifying selection, preserving stable copy numbers regardless of ploidy [38]. Similar patterns have been reported in wheat, where ATG8 expansion is unaffected by species ploidy [39]. The comparatively larger CmATG gene family may be linked to the evolutionary history of C. morifolium. Previous studies indicate that chrysanthemums have undergone multiple whole-genome triplication (WGT) events, along with recent bursts of long terminal repeats (LTRs), resulting in a highly complex and expanded genome structure [27].

Previous studies identified 22 ATG subfamilies in A. thaliana [28] and 24 subfamilies in C. sinensis [30]. A total of 22, 21, and 21 ATG subfamilies were detected in CmATG, CnATG, and ClATG, respectively, with the ATG6 subfamily absent in C. nankingense and the ATG4 subfamily absent in C. lavandulifolium. ATG6 is a core component of the PI3K protein kinase complex, playing a critical role in autophagosome nucleation and vacuolar degradation [40]. On the other hand, ATG4 functions as a cysteine protease that cleaves the C-terminal region of ATG8, enabling it to execute its role in promoting autophagic vesicle formation and autophagosome maturation [18]. These differences suggest that the regulation of autophagy may vary among C. morifolium, C. nankingense, and C. lavandulifolium, potentially reflecting genomic divergence. This finding provides new insights into the evolution and functional adaptation of autophagy-related genes in Asteraceae species. Synteny analysis revealed 252 segmental duplication pairs among CmATG genes, with nearly one CnATG or ClATG gene corresponding to three CmATG genes, suggesting that ATG genes expanded through gene duplication during polyploidization while maintaining high structural conservation.

Cis-regulatory elements are fundamental to the transcriptional regulation of ATG family members. Different transcription factors (TFs) recognize specific cis-elements, enabling precise control over ATG gene expression, thereby influencing autophagic activity and stress resilience in plants. In A. thaliana, the TGA9 transcription factor, a member of the bZIP family, specifically binds to the TGACG (TGA) cis-element located in the promoters of AtATG8B and AtATG8E. The overexpression of AtTGA9 significantly upregulates the expression of these two genes, promoting autophagosome formation and enhancing plant tolerance under nutrient deficiency and abiotic stress conditions such as high salinity and drought [17]. In Chinese flowering cabbage (Brassica rapa ssp. parachinensis), BrMYB108, an R2R3-MYB transcription factor, has been shown to directly interact with the promoters of BrATG5b, BrATG8e-1, and BrATG8h-1. Overexpression of BrMYB108 not only activates autophagy under normal conditions but also enhances autophagic activity under sucrose starvation and osmotic stress, thereby improving plant stress tolerance [41]. WRKY transcription factors are also involved in regulating ATG gene expression by recognizing W-box elements (core sequence: TTGACC/T) within their promoters. For instance, WRKY33 in Arabidopsis interacts with ATG18a during plant defense responses, conferring enhanced resistance against necrotrophic pathogens [42]. Similarly, its homologs SlWRKY33a and SlWRKY33b in tomato are implicated in heat stress responses, where they regulate autophagy-related gene expression to improve thermotolerance [43]. Additionally, the ethylene response factor ERF5 in tomato has been reported to bind to DRE elements (ACCGAC) within the promoters of SlATG8d and SlATG18h, inducing their expression and enhancing autophagic activity under drought conditions, which contributes to increased drought tolerance [44]. In Chrysanthemum, the promoter regions of CmATG family genes are enriched with W-boxes, TGACG motifs, and MYB binding sites, providing potential interaction sites for WRKY, TGA, and MYB transcription factors. The presence of these cis-elements suggests that CmATG genes may exhibit tissue-specific and stress-responsive expression patterns, which could be critical for modulating autophagy under different environmental conditions. This highlights a promising direction for further research into the upstream regulatory networks of CmATG genes, providing insights into their roles in stress adaptation and potential applications in molecular breeding for stress resilience.

Research in A. thaliana has shown that AtATG genes are not tissue-specific [45]. However, the four genes AtATG8a, AtATG8c, AtATG8f, and AtATG8i are widely expressed across various developmental stages and tissues [46]. Similarly, expression analysis in N. tabacum revealed that NtATG genes are present in roots, stems, leaves, pollen, anthers, and seeds, with the highest expression levels observed in pollen and anthers, particularly within the NtATG8 subfamily [33]. Consistent with these findings, most CmATG genes were found to exhibit stable expression across different tissues. However, CmATG8b, CmATG8n, CmATG8d, and CmATG8i showed notably high transcription levels and tissue-specific expression, similar to the patterns observed in Arabidopsis, Nicotiana, and Solanum. The widespread expression of ATG8 subfamily genes in plant tissues suggests their essential role in autophagy throughout plant development and stress responses [34].

The response of CmATG8 genes to heat stress was assessed by analyzing their expression following exposure to 40 °C for 48 h. Members of the CmATG8 subfamily were markedly induced under heat stress, with expression levels generally increasing over time and reaching their maximum at 48 h. Notably, CmATG8o exhibited its highest expression at 72 h. Similar heat-responsive expression patterns have been observed in Capsicum annuum, where most CaATG genes peaked at 1 h of heat treatment, CaATG8c and CaATG18b peaked at 3 h and 6 h, respectively, and nearly all genes returned to baseline levels after 12 h [23]. In Gossypium hirsutum, GhATG8fA showed peak expression at 1 h of heat stress, while GhATG8eD and GhATG8cD reached their highest expression at 24 h [7]. The expression dynamics of CmATG8 genes under heat stress closely resembled those of CaATG8 and GhATG8, suggesting that CmATG8 genes play a crucial role in chrysanthemum’s heat stress response. CmATG8o was identified as a strong candidate gene for future studies on the autophagy-related mechanisms in Chrysanthemum.

This study provides a comprehensive genome-wide characterization of the ATG gene family in Chrysanthemum, including bioinformatic analyses, expression profiling under heat stress, and preliminary functional validation of the CmATG8 subfamily. These findings lay a foundation for future studies on the CmATG gene family and offer valuable genetic resources for breeding heat tolerant chrysanthemum cultivars. Furthermore, the polyploid CmATG gene family analysis serves as a reference for ATG gene studies in other polyploid plant species.

4. Materials and Methods

4.1. Identification of ATG Gene Family Members

The genomic coding sequence (CDS) and protein sequence data, along with annotation files for C. morifolium, C. lavandulifolium, and C. nankingense, were retrieved from the Chrysanthemum Genome Database (http://210.22.121.250:8880/asteraceae/download/downloadPage) (accessed on 4 August 2024). Autophagy-related genes in these species were identified by using the reported A. thaliana ATG protein sequences as references, followed by BLASTp searches across the three genomes performed in TBtools. The search parameters were set with an E-value threshold of <1 × 10⁻⁵ and a sequence similarity of at least 60%.

After removing redundant sequences, the identified candidate ATGs (CmATGs, ClATGs, and CnATGs) were further analyzed for conserved domains using multiple databases, including PFAM (https://pfam.xfam.org/search/) (accessed on 10 August 2024), NCBI-CDD Search (https://plants.ensembl.org/) (accessed on 10 August 2024), and SMART (http://smart.embl-heidelberg.de/) (accessed on 10 August 2024). Only genes containing conserved ATG domains were retained as final ATG gene family members. Additionally, key physicochemical properties of the identified ATG proteins, such as molecular weight and isoelectric point, were predicted using the ExPASy tool (https://web.expasy.org/) (accessed on 12 August 2024).

4.2. Phylogenetic Tree Construction of ATG Gene Family

Multiple sequence alignment of the ATG protein sequences from C. morifolium, C. nankingense, C. lavandulifolium, and Arabidopsis was performed using the Clustal W method in MEGA 11.0. The Neighbor-Joining (NJ) method was used to construct the phylogenetic tree, with a bootstrap value of 1000 replicates.

4.3. Gene Structure, Conserved Motif, and Domain Analysis of ATG Gene Family

Gene structure analysis and visualization were performed using GSDS 2.0 (https://gsds.gao-lab.org/) (accessed on 22 August 2024) based on the coding sequences and full-length sequences of the ATG gene family members. MEME (https://meme-suite.org/meme/tools/meme) (accessed on 25 August 2024) was employed to identify conserved motifs in CmATG proteins, with the motif number set to 20. TBtools was used for visualizing protein domains in combination with the motif data.

4.4. Chromosomal Localization and Synergy Analysis of ATG Gene Family Members

The GFF3 annotation files for each genome were imported into TBtools. The IDs of the identified ATG genes were input, and a map depicting the distribution and localization on the chromosomes of Chrysanthemum was constructed. Synteny relationships of ATG genes between C. morifolium, C. nankingense, and C. lavandulifolium was conducted using MCScanX in TBtools. The results were visualized using TBtools’ Advanced Circos and Multiple Synteny Plot.

4.5. Analysis of Cis-Regulatory Elements in the Promoters of the Chrysanthemum ATG Gene Family

The 2000 bp upstream of the start codon of each ATG gene family member was extracted from the respective genomes and submitted to PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 27 August 2024) for prediction of cis-acting elements. The results were compiled and visualized using TBtools.

4.6. Tissue-Specific Expression of the Chrysanthemum ATG Gene Family

Expression data for all identified CmATG genes in different tissues were retrieved from the Chrysanthemum transcriptome database (http://210.22.121.250:8880/asteraceae/rna/searchPage) (accessed on 30 August 2024). The expression levels of these genes in various tissues were imported into TBtools to generate a heatmap.

4.7. Expression Analysis of CmATG8 Subfamily in Response to Heat Stress

Chrysanthemum cultivar ‘Jinba’ tissue culture plantlets were used as experimental materials. After transplanting into pots, the plantlets were grown in a controlled growth chamber under the following conditions: 25 °C/22 °C (day/night), 5000 lux light intensity, and a 14 h/10 h light/dark photoperiod. When the plants developed 8–10 fully expanded leaves, they were subjected to heat stress at 40 °C. Leaf samples were collected at 0, 1, 3, 6, 12, 24, 48, and 72 h after treatment. Total RNA was extracted from the leaves and reverse transcribed into cDNA. Gene specific primers (Supplementary Table S4) were designed for qRT-PCR analysis. CmEF1α was used as the internal reference gene. Prior to conducting inferential tests, data normality was assessed using the Shapiro–Wilk test, and homogeneity of variances was evaluated with Levene’s test in IBM SPSS 22.0. Relative gene expression levels were calculated using the 2^−ΔΔCt method. Data meeting the assumptions of normality and equal variances were analyzed by one-way ANOVA followed by Duncan’s multiple range test at a significance level of p < 0.05 using DPS 9.01. Bar graphs were generated with Origin 2022. Results are presented as mean ± SD.

5. Conclusions

A total of 130, 51, and 49 ATG genes were identified in C. morifolium, C. nankingense, and C. lavandulifolium, with uneven chromosomal distribution. Collinearity analysis indicated a closer evolutionary relationship between C. morifolium and C. lavandulifolium, while phylogenetic and conserved motif analyses revealed subfamily-specific conservation and structural diversity. Transcriptomic profiling showed broad expression of CmATG genes, with floral buds exhibiting the highest levels and strong heat-induced upregulation in the ATG8 subfamily, particularly CmATG8o.

The identification of heat-responsive CmATG genes, particularly within the ATG8 subfamily, provides promising genetic targets for enhancing heat tolerance in chrysanthemum through molecular breeding or genome-editing. However, the functional roles of ATG proteins in autophagosome biogenesis, protein–protein interactions, and metabolic reprogramming under stress remain unclear. Future studies should prioritize functional validation of key genes and elucidation of the associated signaling pathways and regulatory networks.