Inhibition of Autophagy Increases Freezing Survival in Arabidopsis thaliana

Inhibition of Autophagy Increases Freezing Survival in Arabidopsis thaliana | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Inhibition of Autophagy Increases Freezing Survival in Arabidopsis thaliana Yushi Peng, Shujuan Guo, Ben Lei, Qiuling Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6620529/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Jul, 2025 Read the published version in BMC Plant Biology → Version 1 posted 4 You are reading this latest preprint version Abstract Background Plants have evolved multiple strategies to cope with the ever-changing external environment. Autophagy, as one of the crucial mechanisms involved, has been demonstrated to play a pivotal role in plant responses and adaptation to abiotic stresses. However, the precise molecular mechanisms underlying the role of autophagy in mediating cold stress remain to be fully elucidated. Results In this study, we demonstrated that autophagy mutants presented increased freezing tolerance under both non-acclimated and cold-acclimated conditions. Autophagy positively regulates the expression of anthocyanin biosynthesis-related genes, thereby influencing anthocyanin accumulation in Arabidopsis under low-temperature conditions. Moreover, we found that cold stress directly suppresses the expression of autophagy-related genes and reduces autophagic flux. The RNA-seq data revealed that cold-responsive genes were pre-activated in the autophagy mutant atg13ab even before cold treatment. Additionally, we observed constitutive accumulation of the dehydrin protein COR47 in atg13ab . Conclusions Taken together, these data suggest that autophagy is a negative regulator of freezing tolerance in Arabidopsis. Autophagy Arabidopsis cold stress atg13ab ATG8 COR47 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Plants face dual challenges of biotic and abiotic stresses at different growth stages. Cold stress, a critical environmental factor limiting plant geographical distribution and agricultural production, significantly inhibits plant growth and development by disrupting cell membrane integrity, inducing oxidative stress and interfering with metabolic homeostasis [ 1 – 3 ]. Cold stress triggers the abnormal accumulation of denatured substances (such as misfolded proteins and damaged organelles) in plant cells [ 4 ]. If these abnormal components are not promptly cleared, they form toxic aggregates, disrupt cellular metabolism, inhibit growth and development, and weaken stress resistance [ 5 ]. To address the clearance of damaged proteins under stress conditions, plants have evolved two classical degradation pathways: the autophagy-dependent degradation pathway and the ubiquitin-dependent 26S proteasome degradation pathway [ 6 , 7 ]. The ubiquitin‒proteasome system (UPS) primarily degrades soluble small-molecule proteins and peptides tagged with polyubiquitin chains, while autophagy is responsible for clearing insoluble protein aggregates, large protein complexes, intracellular pathogens, and damaged organelles [ 6 , 8 ]. The synergistic action of these two pathways forms the core framework of the plant protein quality control network. The autophagy process involves a membrane-independent extension structure that can encapsulate cytoplasmic components [ 9 , 10 ]. This process relies on the coordinated action of various ATG (AuTophaGy-related) proteins. In plants, at least 36 ATG proteins have been identified, forming different complexes that regulate various stages of autophagy: initiation, vesicle nucleation, elongation, maturation, fusion of autophagosomes with vacuoles, and degradation of autophagosomes [ 11 – 13 ]. Among these, the ATG1 kinase complex (ATG1-ATG13-ATG11-ATG101) is responsible for sensing the nutritional status of cells and initiating autophagy when needed. The PI3K (phosphatidylinositol 3-kinase) complex (VPS34-VPS15-ATG6-ATG14) catalyzes the production of specific lipid signals, PI3P (phosphatidylinositol 3-phosphate), aiding in the remodeling of autophagosome membrane structures. The ATG9 complex (ATG9-ATG2-ATG18) primarily mediates lipid transport, supporting the expansion of autophagic vesicles. The two ubiquitin-like systems, ATG8-PE (phosphatidylethanolamine) and ATG5-ATG12, participate in the maturation of autophagosomes, preparing them for sealing. The SNARE (soluble NSF attachment protein receptor) complex regulates the membrane fusion of autophagosomes with vacuoles, ensuring the efficient degradation of substrates. These systems work in concert to complete the entire process of autophagy, from induction to degradation [ 11 ]. Autophagy normally maintains basal levels of activity in plants [ 14 – 16 ] but is induced under nutrient-deficient conditions, including nitrogen, carbon, phosphorus, and sulfur [ 17 – 21 ]. Additionally, autophagy also serves as a critical mechanism for plants to cope with abiotic stresses, with the majority of autophagy mutants showing increased sensitivity to salt stress [ 22 , 23 ], drought stress [ 24 ], hypoxia stress [ 25 ], and heat stress [ 26 , 27 ], highlighting the role of autophagy in adaptation to abiotic stresses. Current evidence regarding the role of plant autophagy in the cold response remains contradictory. A previous study revealed that the accumulation of autophagosomes increases and that the expression of most CaATG genes is induced in Capsicum annuum upon cold treatment [ 28 ], indicating that autophagy may positively regulate the plant cold stress response. Moreover, chilling stress significantly induces the transcription of a series of autophagy-related genes, such as ATG2 , ATG6 , and ATG8 , and enhances the autophagic flux in tomato. Increased autophagic activity thus improves the cold tolerance of plants [ 29 ]. Further research revealed that among this series of cold-induced tomato ATG genes, ATG18a is also included. The mutant of ATG18a exhibited sensitivity to cold treatment, along with decreased autophagosome formation and increased accumulation of ubiquitinated proteins [ 30 ]. Cold also induces the expression of selective autophagy receptors such as NBR1a (a neighbor of BRCA1), NBR1b , and SEC62 in tomato, and silencing these selective autophagy receptors affects the generation of autophagy, leading to compromised cold tolerance [ 31 ]. Additionally, ATG13 is upregulated in response to cold stress in Medicago sativa , and compared with wild-type plants, transgenic tobacco overexpressing MsATG13 enhanced cold tolerance compared to wild-type plants due to the upregulation of other ATGs that are necessary for autophagosome production under cold stress conditions [ 32 ], suggesting the positive role of autophagy in plant resistance to cold stress. However, some studies indicate that autophagy in plants is restricted under low-temperature conditions. Compared with that in normal plants, the transcription of autophagy-related genes in cold-treated Arabidopsis was significantly suppressed. The levels of the autophagy marker protein ATG8 decreased substantially, whereas the protein accumulation of the autophagy substrate NBR1 increased markedly [ 33 , 34 ]. These observations suggest that autophagy is inhibited or impaired under cold stress. A recent study revealed that autophagy is dispensable for cold acclimation and freezing tolerance in Arabidopsis, atg5-1 and atg10-1 mutant exhibited normal freezing tolerance, cold-regulated gene expression showed no significant differences between the atg mutants and wild- type as well as autophagy was rarely induced by cold exposure [ 35 ]. These conflicting results suggest the complex role of autophagy in plant cold stress responses, as well as the functional divergence of autophagy across species under stress conditions. In this study, we found that autophagy mutants of Arabidopsis exhibited enhanced tolerance to freezing. Autophagy was significantly suppressed by cold, both at the transcriptional and protein levels. Additionally, cold-responsive genes were constitutively activated in the autophagy mutants, and the cold-regulated protein COR47 was more stable in the autophagy defect mutants. These findings reveal that autophagy serves as a negative regulatory pathway for plant adaptation to cold stress. Results Autophagy negatively regulates freezing tolerance in Arabidopsis To explore the contributions of autophagy to freezing tolerance in Arabidopsis, various autophagy mutants ( atg1abct , atg13ab , atg5-1 , and atg7-3 ) were collected as described previously [ 19 , 36 ]. Fourteen-day-old seedlings were subjected to freezing treatment. Under cold-acclimated (CA) conditions, these autophagy mutants exhibited stronger freezing tolerance compared to the wild type (reflected by higher chlorophyll content) (Fig. 1 A-C). Next, we examined ion leakage, which is an indicator of stress-induced plasma membrane damage, in the autophagy mutants. Ion leakage in the mutants under CA conditions was consistently lower than that in the wild type (Fig. 1 D, E). Since autophagy is implicated in plant stress memory, autophagy mutants that undergo heat priming retain thermotolerance memory and exhibit increased resistance to subsequent heat stress [ 27 ]. We therefore investigated whether non-acclimated (NA) autophagy mutants maintained improved freezing tolerance. Consistent with their performance under CA conditions, these autophagy mutants still displayed higher chlorophyll contents and lower ion leakage rates without cold acclimation (Fig. 1 A-E). Furthermore, proline, a well-documented stress-protective metabolite in plants [ 37 ], was found to accumulate at higher levels in autophagy mutants than in wild-type plants following cold treatment (Fig. 1 F). These findings collectively demonstrate that autophagy serves as a negative regulator of freezing tolerance in Arabidopsis. Autophagy regulates the accumulation of anthocyanins in Arabidopsis under chilling conditions To validate the function of autophagy under chilling conditions in plants, the wild type and all the autophagy mutants were grown at 22°C and 4°C under normal photoperiod conditions (16 h/light and 8 h/dark), respectively. No significant differences were detected among the plants grown at 22°C (Fig. 2 A). However, under continuous growth at 4°C for 45 days, the wild-type plants exhibited a noticeable accumulation of anthocyanins in their cotyledons, which was consistent with the findings of a previous study [ 38 ]. However, the cotyledons of the mutants still maintained a distinct green color (Fig. 2 A, B). As DFR (dihydroflavonol 4-reductase), CHS (chalcone synthase), and ANS (anthocyanin synthase) are key enzymes in anthocyanin biosynthesis [ 39 , 40 ], further analysis of the expression of these genes in different plants revealed that the expression of DFR , CHS , and ANS was significantly suppressed in the autophagy mutants atg13ab and atg5-1 both before and after cold treatment (Fig. 2 C). This finding is consistent with a previous study showing down-regulation of the flavonoid biosynthetic pathway in autophagy mutants [ 41 ]. These results indicate that autophagy negatively regulates anthocyanin accumulation in Arabidopsis under chilling conditions and that anthocyanins likely do not directly contribute to the increased cold tolerance observed in autophagy mutants. Cold inhibits the expression of autophagy-related genes To further investigate how cold affects autophagy in Arabidopsis, the transcription of autophagy-related genes were first examined after treatment at 4°C under normal photoperiod conditions. It was found that cold treatment significantly suppressed the expression of a series of autophagy-related genes, such as ATG8a , ATG8e , ATG1a , ATG13a , ATG5 , and ATG7 (Fig. 3 A), which is consistent with previous reports [ 33 ]. To further characterize the transcriptional regulation of autophagy-related genes, we generated stable transgenic lines by fusing the 2000-bp promoter regions upstream of the ATG8a and ATG13a transcription start sites to the β-glucuronidase (GUS) reporter gene. Histochemical GUS staining revealed that under normal growth conditions, both the ATG8a and ATG13a promoters drove strong reporter gene expression in the cotyledons and roots of Arabidopsis seedlings. However, following cold treatment at 4°C, we observed a significant reduction in GUS expression for both genes after 6 hours of cold exposure, and this suppression was particularly pronounced in cotyledons. The inhibitory effect became more evident after 24 hours of cold treatment (Fig. 3 B). These results indicate that low temperatures can inhibit the transcription of autophagy genes in Arabidopsis. Autophagic flux is reduced upon cold treatment The suppression of autophagy gene expression by cold treatment suggests that autophagic activity may also be inhibited after cold treatment. To validate this hypothesis, the pATG8a:GFP-ATG8a /Col transgenic line, a well-characterized autophagosome marker line [ 42 ], was used. In the presence of concanamycin A (ConA), an autophagy inhibitor that stabilizes autophagic bodies [ 17 , 18 ], it was observed that the number of autophagosomes labeled with GFP-ATG8a significantly decreased after cold treatment (Fig. 4 A, B). We then used the release of free GFP from GFP-ATG8a to monitor autophagic transport, as previously reported [ 18 ]. Consistent with the microscopy results, free GFP levels were substantially reduced upon cold treatment (Fig. 4 C, D). The lipidation of ATG8 and the cleavage efficiency of the GFP-ATG8 fusion protein are commonly used indicators of autophagic flux [ 18 , 43 ]. Consistent with previous findings, both lipidated and non-lipidated ATG8 rapidly decreased following cold treatment [ 34 ] (Fig. 4 E, F). These results confirm that cold inhibits autophagic flux in Arabidopsis. Differentially expressed gene (DEG) analysis between Col and the atg13ab mutant To further investigate the effect of autophagy on the transcriptome profile, we performed RNA-seq analysis of 14-day-old wild-type and atg13ab seedlings treated at 4°C for 0, 3, or 24 h. Two independent experiments were carried out (Fig. 5 A), and the differentially expressed genes were analyzed via HTSeq and DESeq2 software. At 3 h after cold treatment, 959 genes were up-regulated (log2 ≥ 1, FDR ≤ 0.01), and 623 genes were down-regulated (log2 ≤ 1, FDR ≤ 0.01) in the wild-type. When cold treatment was extended to 24 h, 2163 genes were up-regulated and 2086 genes were down-regulated (Fig. 5 B). In the atg13ab mutant, the up-regulated genes numbered 870 and 2296, respectively, at 3 h or 24 h of cold treatment, and the down-regulated genes numbered 1090 and 2367, respectively (Fig. 5 B). Moreover, a total of 1999 cold-induced genes and 1706 cold-repressed genes were identified in both the wild-type and atg13ab mutant (Fig. 5 C, 5 D). These results suggest that a large number of cold regulate genes are not affected in the atg13ab mutant under cold stress. For both Col and atg13ab , the number of DEGs induced at 24 h was significantly greater than that induced at 3 h (Fig. 5 E), indicating that cold treatment for 24 h caused substantial transcriptional rearrangement within the plant. Additionally, it was observed that a large number of DEGs were found in both wild-type and atg13ab even under normal growth conditions, while the number of DEGs in Col and atg13ab decreased as the duration of cold treatment extended (Fig. 5 E). Compared with Col, untreated atg13ab had 525 DEGs, with 337 genes up-regulated and 148 genes down-regulated; at 3 h of cold treatment, there were a total of 272 DEGs in Col and atg13ab , with 181 upregulated and 91 downregulated; and at 24 h of cold treatment, these DEGs numbered only 161, including 37 up-regulated and 124 down-regulated (Fig. 5 E). These results indicated that cold stress greatly reduced the difference in gene expression profiles between Col and the atg13ab mutant, suggesting that autophagy deficiency also has a substantial effect on gene expression but that this effect occurs primarily in untreated plants. GO analysis of DEGs between unstressed Col and the atg13ab mutant The wild-type and atg13ab mutant displayed a large difference in gene expression profiles prior to cold treatment (Fig. 5 E). To understand the effects of autophagy on plant gene expression under normal temperature, we first performed GO enrichment analysis to assign biological processes to the identified DEGs between Col and atg13ab at 0 h of cold treatment (Fig. 6 A, B). Among the up-regulated DEGs in the atg13ab mutant plants, those involved in transcription and DNA replication, the stress response, the organonitrogen compound and acid chemical response, and the osmotic stress response were substantially enriched (Fig. 6 A). These findings suggest that autophagy deficiency may lead to dysregulation of the cell cycle and that intracellular nitrogen metabolism, the cytoplasmic pH, and osmotic pressure may be altered. Surprisingly, we found that genes responsive to cold and freezing were also enriched (Fig. 6 A), including early cold-responsive genes such as the CBF (C-repeat binding factor)/DERB1 (dehydration-responsive element binding) transcription factors CBF1 , CBF2 , and DDF1 (dwarf and delayed flowering 1) [ 44 , 45 ], along with well-characterized cold-inducible genes such as B1L1 (BYPASS1-LIKE), ZAT12 (ZINC FINGER OF ARABIDOPSIS THALIANA12), and MYB15 [ 46 – 48 ] (Fig. 6 C), which is consistent with previously reported constitutive upregulation of the cold-responsive genes CBF1 , CBF2 , MYB15 , and ZAT12 at 60 DAS (days after sowing), which normally occurs in the atg5 mutant compared with the wild type [ 41 ], indicating that the cold signaling pathway is constitutively activated in the atg13ab mutant under basal conditions. This may be the primary reason for the tolerance of the autophagy mutant to cold stress. Further analysis of the down-regulated DEGs in Col and atg13ab under unstressed conditions revealed that these genes were enriched primarily in pathways related to single-organism metabolism, cellular protein modification, hydrogen peroxide metabolism, cellular detoxification, and drug transport (Fig. 6 B). This suggests that the mutant has altered basal metabolism, disrupted protein homeostasis, impaired oxidative stress management, and reduced detoxification capacity and xenobiotic resistance. We also observed that genes involved in flavonoid biosynthesis were downregulated in the mutant, including the aforementioned anthocyanin biosynthesis-related genes ANS and DFR , as well as the key anthocyanin regulatory transcription factors MYB75 and NAC042 [ 49 , 50 ] (Fig. 6 D). These findings are consistent with our qRT-PCR results from previous chilling experiments and earlier reports demonstrating the constitutive downregulation of anthocyanin-related genes in the autophagy mutant atg5 [ 41 ]. Additionally, the plant defense response and incompatible interaction-related genes were down-regulated in the mutant (Fig. 6 B), suggesting that the mutant may have defects in plant immunity. GO analysis of DEGs between Col and the atg13ab mutant in response to cold stress As described previously, cold triggers extensive transcriptional reprogramming in Arabidopsis (Fig. 5 ). To systematically investigate how cold affects plant gene expression, we performed GO enrichment analysis of DEGs in Col plants under cold treatment. Our analysis revealed that after 3 h and 24 h of cold exposure, the up-regulated DEGs were enriched predominantly in pathways related to transcription; DNA replication; organonitrogen compound, acid chemical, and metal ion responses; cold and osmotic stress response; and RNA processing, or metabolism (Supplementary Fig. 1A). Conversely, the down-regulated DEGs were enriched primarily in pathways such as transcription, DNA replication, single-organism metabolism, protein modification, hormone responses, and lipid/fatty acid metabolism (Supplementary Fig. 1B). Notably, we observed cold-induced up-regulation of salicylic acid-related and fungal defense-related genes in Col (Supplementary Fig. 1A), which is consistent with previous reports showing that cold activates immunity-related pathways [ 51 ]. We further analyzed the DEGs between Col and atg13ab mutants after cold treatment. The up-regulated genes were enriched mainly in processes related to the stress response; transcription and DNA replication; acid, chemical, hexose, and disaccharide responses; and lipid or protein localization (Fig. 7 A, B). These suggest that autophagy defects may lead to increased cellular stress, thereby activating stress responses and DNA repair mechanisms. Additionally, altered sugar metabolism could provide energy to sustain cell survival under cold conditions, whereas changes in lipid localization might help maintain membrane fluidity and prevent cold-induced membrane damage. Furthermore, a small number of cold-responsive genes remained up-regulated in the mutant even after 24 h (Fig. 7 B), which may represent potential mechanisms for cold stress tolerance in the atg13ab mutant. Surprisingly, although a small number of immune pathway-related genes were already down-regulated in atg13ab under untreated conditions (Fig. 6 B), a large proportion of down-regulated DEGs after 3 h and 24 h of cold treatment clustered in bacterial and fungal defense response pathways (Fig. 7 C, D). These indicate that the atg13ab mutant may suffer from immune deficiency, which compromises its ability to respond to pathogen threats during cold stress. To confirm the RNA-seq data, cold-associated genes, including CBF1/2/3 , COR15a (COLD-REGULATED 15a), COR47 , Gols3 (galactinol synthase 3), DDF1 , B1L , and ZAT12 , were selected for qRT-PCR [ 52 ]. The expression of the CBF genes DDF1 , B1L , and ZAT12 was elevated in the mutant under unstressed conditions, which was consistent with the RNA-seq results (Fig. 8 ). After 3 h of cold stress, CBFs , DDF1 , B1L , and ZAT12 in atg13ab still maintained high expression levels‌. Although the basal expression of late cold-responsive genes ( COR15a , COR47 , Glos3 ) was comparable to that of the wild type under normal conditions, their induction in the mutants was significantly greater than that in the wild type after 24 hours of cold treatment‌ (Fig. 8 ). These results reveal that autophagy deficiency leads to persistent activation of the cold signaling pathway in plants‌. COR47 accumulated in the autophagy mutant atg13ab Given the marked up-regulation of cold-responsive genes in the mutants, we hypothesized that the proteins encoded by these genes might also accumulate at relatively high levels in these lines. Previous proteomic analyses of Col and the autophagy mutants atg1abct , atg11 , and atg5 revealed elevated COR47 expression in the mutant background [ 53 , 54 ]. To further investigate the stability of COR47 in the atg13ab mutant, MBP-COR47 was purified in vitro, and its stability was examined with cell-free analysis. The results revealed that the degradation rate of MBP-COR47 was obviously inhibited in the mutant (Fig. 9 A, B). Furthermore, the COR47-GFP fusion protein was transiently expressed in the protoplasts of Col and the atg13ab mutant. At 22°C, the COR47 protein was expressed at low levels in the wild-type plants but was significantly elevated in the atg13ab mutant plants (Fig. 9 C, D). With prolonged cold treatment, rapid induction of COR47 expression was observed in Col, while the atg13ab mutant exhibited even greater accumulation (Fig. 9 E, F). These results suggest the constitutive accumulation of COR47 in the autophagy-deficient atg13ab mutant, suggesting that COR47 may be a potential substrate of autophagy. To further investigate the relationship between COR47 and autophagy, we first analyzed its subcellular localization and found that COR47 primarily localizes to the cytoplasm rather than the nucleus (Supplementary Fig. 2), which is consistent with the findings of a previous study [ 55 ]. However, neither yeast two-hybrid nor BiFC assays detected any interaction between COR47 and core autophagy-related proteins, although COR47 has been shown to form homodimers [ 55 ] (Supplementary Fig. 3). Therefore, COR47 likely regulates its stability through mechanisms independent of direct interactions with core autophagy proteins. Discussion Autophagy, as a protective strategy, has been demonstrated to play a positive role in plant responses to multiple stresses [ 56 , 57 ]. However, in this study, we revealed a previously unrecognized negative regulatory role of autophagy in Arabidopsis freezing tolerance independent of cold acclimation (Fig. 1 ). To elucidate the mechanism underlying the enhanced cold tolerance of the autophagy-deficient plants, we analyzed protective metabolites, including proline and anthocyanins. Notably, compared with wild-type plants, autophagy mutants accumulated higher levels of proline after cold treatment (Fig. 1 F), whereas anthocyanin accumulation was significantly lower in these mutants. These findings suggest that anthocyanins likely do not directly contribute to the cold-tolerant phenotype observed in autophagy mutants. Unlike previous reports attributing reduced anthocyanin levels in autophagy mutants to impaired vacuolar trafficking [ 58 ], our transcriptomic and qRT-PCR data demonstrated that anthocyanin biosynthesis-related genes (e.g., DFR , ANS , CHS ) were constitutively downregulated in the atg13ab and atg5-1 mutants under basal conditions, which is consistent with previous observations [ 41 ]. These results imply that autophagy may suppress anthocyanin production by directly inhibiting the flavonoid biosynthetic pathway rather than affecting vacuolar transport. Nevertheless, the molecular mechanisms by which autophagy downregulates flavonoid metabolism remain unclear and warrant further investigation. To better understand the mechanistic contributions of autophagy to cold stress responses in Arabidopsis, we examined the effects of cold stress on autophagy and observed significant suppression of autophagic activity, which is consistent with previous studies [ 33 , 34 ]. This inhibition may stem from plants redirecting energy resources under cold stress: rather than fueling the highly energy-demanding catabolic process of autophagy, limited ATP is prioritized for the rapid synthesis of cold-adaptive enzymes, metabolites, and cryoprotective compounds (e.g., proline, soluble sugars), as well as activation of the CBF/DREB signaling pathway. However, the precise molecular mechanisms underlying cold-induced autophagy suppression remain unclear and represent an important direction for future research. Several potential mechanisms warrant consideration: (1) Cold stress might induce repressive chromatin states (e.g., H3K27me3 deposition) at autophagy gene promoters through vernalization-like epigenetic mechanisms, as autophagic genes are known to undergo dynamic epigenetic modifications [ 59 ]; (2) reduced lipid unsaturation at low temperatures could impair membrane fluidity [ 60 ], potentially disrupting interactions between autophagic precursors (e.g., phagophores) and PI3P-enriched domains, thereby hindering autophagosome assembly; and (3) in Jatropha curcas seedlings, cold treatment markedly increased the phosphorylation levels of ATG13 [ 61 ], suggesting that unidentified cold-activated kinases may phosphorylate autophagic components, altering their stability or function to suppress autophagic flux. To elucidate the broad role of autophagy in plant cold tolerance, we conducted comparative transcriptome analysis of wild-type Arabidopsis and autophagy-deficient atg13ab mutants. We observed that even under nonstress conditions, Col and atg13ab already exhibited substantial differences in gene expression, including the expression of cold-responsive genes such as CBFs , DDF1 , B1L , and ZAT12 , which is consistent with previously reported constitutive upregulation of the cold-responsive gene atg5 [ 41 ]. This may suggest that autophagy deficiency places cells in a persistent state of stress, leading to constitutive activation of the CBF/DREB1 signaling pathway even in the absence of external cold stress. The pre-activation of CBF signaling in autophagy mutants mimics the “cold priming” phenomenon [ 62 ], allowing rapid deployment of cryoprotective mechanisms without prior cold exposure. However, the precise mechanism by which these genes are activated in the mutants remains unclear. One possible explanation is that certain positive regulators of the cold response, such as transcription factors (e.g., ICE1) or kinases (e.g., OST1), may serve as autophagy substrates. The accumulation of these genes in autophagy-deficient mutants could indirectly increase the transcriptional activity of CBF and related genes. Notably, in atg13ab , genes associated with immunity against bacteria, fungi, and oomycetes were downregulated before and after cold treatment. Extensive studies have demonstrated that cold stress triggers a defense-like response similar to pathogen invasion, which typically leads to elevated salicylic acid (SA) levels and the activation of defense-related genes to prepare plants for sensitization to future pathogen infection [ 63 ]. However, immune responses are energetically costly and may constrain plant growth under cold conditions [ 64 ]. The observed attenuation of cold-induced immune signaling in atg13ab indicates that ATG13 may prevent overactivation of these pathways under non-pathogenic conditions to avoid autoimmunity, thereby increasing the freezing tolerance of the mutant. Dehydrins play crucial roles in plant responses to water deficit and are considered to contribute to the protection of fragile organellar structures under adverse conditions [ 65 , 66 ]. Although drought and freezing are distinct stresses, freezing-induced cellular dehydration triggers substantial accumulation of dehydrins [ 67 ]. Through cell-free and protoplast expression assays, we found that the COR47 protein was strongly induced by cold in wild-type plants, whereas it exhibited abnormal accumulation in autophagy mutants even under normal temperatures. This finding further explains the enhanced freezing tolerance phenotype of the autophagy-deficient mutants. A comparative proteomic analysis of wild-type and atg1abct mutants under drought stress revealed significant enrichment of cold-regulated differentially expressed proteins (DEPs), including COR47, RESPONSIVE TO DESICCATION 29A (RD29A), and LOW TEMPERATURE-INDUCED 30 (LTI30), in the atg1abct mutant prior to drought treatment [ 53 ]. Furthermore, studies in atg11 and atg5 mutants demonstrated that COR47 displayed higher protein abundance and lower degradation rates than did the wild type [ 54 ]. These observations suggest a potential association between the dehydrin COR47 and autophagy, as previously described [ 68 ]. However, we did not detect any direct interaction between COR47 and core autophagy proteins. This finding implies the possible existence of a specific autophagy receptor that targets COR47 as a cargo for degradation via the autophagy pathway. Although we have uncovered that autophagy negatively regulates freezing stress tolerance in Arabidopsis , this finding contrasts with the well-established protective role of autophagy in cold resistance among Solanaceae crops such as tomato and pepper [ 28 , 29 , 31 ], as silencing core ATG genes in tomato compromises chilling resistance by impairing the clearance of ubiquitinated protein aggregates [ 30 ]. The mechanistic divergence in autophagy-mediated temperature adaptation across species remains unresolved, potentially reflecting evolutionary divergence in climate adaptation strategies between cold-adapted plants (e.g., Arabidopsis) and temperate crops (e.g., tomato and pepper). Future investigations encompassing a broader phylogenetic spectrum of plant species will be critical for revealing the functional diversity of autophagy in plant cold adaptation. In summary, our study demonstrated that autophagy functions as a negative regulator in Arabidopsis in response to cold stress. This contrasts with its well-documented protective roles in other stress conditions, highlighting autophagy as a “double-edged sword” in plant stress adaptation. The complex biological outcomes of autophagy, whether promoting or suppressing cell death under stress, depend on intricate contextual factors that warrant further investigation. Materials and methods Plant materials and growth conditions All materials used in this work were in the Arabidopsis thaliana accession Columbia (Col-0) background. Mutants of atg5-1 (SAIL_129_B07) and atg7-3 (SAIL_11_H07) were obtained from the Arabidopsis Biological Resource Center (ABRC). atg13ab (SALK_044831) and atg1abct were obtained from Prof. Fa Qiang Li [ 19 , 36 ]. Other stable expression transgenic plants were generated in this study. Arabidopsis seedlings were surface sterilized and vernalized at 4℃ for 2 to 3 days, then germinated on 1/2 MS medium at 22℃ under an LD photoperiod (16 h light/8 h darkness) with illumination at ~ 100 µmol m − 2 s−1 . After 1 week, the seedlings were transferred to soil for further growth. Nicotiana benthamiana was subsequently grown under LD conditions. One-month-old tobacco plants were used for transient expression assays. Assays for freezing tolerance, chlorophyll content, electrolyte leakage, and proline content Freezing tolerance was assessed as previously described [ 69 ]. Briefly, 10-day-old plants grown at 22°C on 1/2 MS plates were treated with or without cold acclimation (4°C for 3 d) and then subjected to a freezing assay. The program was set at 0°C, and 1°C h was dropped to the desired temperature. After freezing treatment, the plants were incubated at 4°C in the dark for 12 h and then transferred to 22°C for an additional 2–3 d. Chlorophyll was extracted and measured as described [ 70 ]. Electrolyte leakage assays were performed as described [ 69 ]. The proline content was measured as previously described [ 45 ]. At least three independent experiments were performed, and each experiment was performed with three technical replicates. Chilling stress response assay and anthocyanin extraction For chilling stress, 7-day-old wild-type, atg1abct , atg13ab , atg7-3 , and atg5-1 seedlings grown on 1/2 MS plates at 22°C were transferred to a 4°C growth chamber (16 h light/8 h darkness with illumination at ~ 100 µmol m − 2 s−1 ) and maintained for the indicated times. At least three independent experiments were performed, and each experiment was performed with three technical replicates. Anthocyanin measurement was performed as described previously [ 40 ]. Arabidopsis seedlings were incubated in extraction buffer (methanol containing 1% HCl) overnight at 4°C in the dark. The samples were centrifuged, and the supernatants were collected for absorbance quantification at 530 and 657 nm. (A530 − 0.25 × A657) per gram fresh weight was used to quantify the relative amounts of anthocyanins. qRT-PCR analysis RNA extraction was performed according to the instructions of the total RNA kit (OMEGA). 5 × PrimeScript™ RT Master Mix (TAKARA) was used to synthesize cDNA. qRT-PCR analysis was performed using SYBR Premix Ex Taq II (TaKaRa) and a StepOne PCR instrument. The 2− △△ CT method was used to calculate the relative expression of genes. Ubiquitin 10 ( UBQ10 ) was used as a reference gene. The gene-specific primers used for qRT-PCR are listed in Supplemental Table 1. GUS staining Transgenic plants harboring the proATG13a::GUS or proATG8e::GUS constructs were immediately immersed in GUS staining solution (10 mM EDTA disodium salt, 100 mM NaH 2 PO 4 , 0.5 mM K 4 Fe(CN) 6 , 0.5 mM K 3 Fe(CN) 6 , 0.5 mg/ml X-gluc (B5285, Sigma), pH 7.0) after various durations of 4°C treatment, incubated for 5 h at 37°C, and then decolorized with 70% ethanol. The stained tissues were observed and photographed with a Zeiss Discover.v20 imaging system. Drug treatment and confocal laser scanning microscopy For concanavalin A (ConA) treatment, seedlings of proATG8a::GFP-ATG8a /Col were first grown on 1/2 MS agar media for 5 days at 22°C. The sterile seedlings were then transferred to 1/2 MS liquid media and either continued culturing at 22°C for 12 h (control) or exposed to 4°C in a growth chamber for 12 h under a standard photoperiod (16 h light/8 h dark cycle with ~ 100 µmol m⁻² s⁻¹ light intensity). After treatment, the seedlings were allowed to recover for 6 h at the normal temperature (22°C). Subsequently, either DMSO (control) or 0.5 µM ConA was added to the liquid medium, followed by further incubation at 22°C for 4 h. Following incubation, the GFP labeled autophagosomes in the roots were visualized with a Nikon A1 + confocal laser scanning microscopeusing 40 × water objectives. Excitation was performed at 488 nm, emission was collected at 500–530 nm. Three to four representative images in the root elongation zone were photographed per seedling, and the number of GFP-ATG8a puncta in each image was counted and averaged. A total of ten seedlings were observed per treatment. Protein isolation and immunoblot analysis For protein extraction, Arabidopsis samples were ground and homogenized in ice-cold extraction buffer (10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM EDTA pH 8.0; 10% Glycerol; 0.5% Triton X-100; 1 × cocktail). Samples were incubated on ice for 10–15 min and centrifuged at 4℃ for 10 min at 12,000 g . The supernatant was used for electrophoresis. For immunoblot analysis, total proteins were subjected to SDS-PAGE and electrophoretically transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore). For ATG8a assay, total protein was separated by SDS-PAGE in the presence of 6 M urea. The antibodies used for protein blot analysis were against ATG8a (Abcam, ab77003, 1:1000), GFP (Abmart, M20004, 1:5000), and β-actin (CWBIO, CW0264, 1:5000). High-throughput mRNA sequencing analysis Fourteen-day-old seedlings grown on 1/2 MS medium at 22°C were treated at 4°C for 0, 3 or 24 h. Total RNA was extracted. and 3 µg of RNA for each sample was used for library construction and subsequent RNA-deep sequencing on the Illumina HiSeq 2500 platform. RNA-Seq data were collected from two independent experiments. The adaptor sequences and low-quality sequence reads were removed from the data sets. Raw sequences were transformed into clean reads after data processing. These clean reads were then mapped to the reference genome sequence. Only reads with a perfect match or one mismatch were further analyzed and annotated on the basis of the reference genome. HISAT2 tools were used for mapping with the reference genome. Gene function was annotated on the basis of TAIR10. Differential expression analysis of two conditions/groups was performed via the DESeq2 R package (1.26.0). DESeq2 provides statistical routines for determining differential expression in digital gene expression data via a model based on the negative binomial distribution. The resulting P values were adjusted via Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with FDR < 0.05 & |Log2(foldchange)| ≥1 found by DESeq2 were considered differentially expressed. Gene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) was implemented via the GOseq R package-based Wallenius non-central hyper-geometric distribution (Young et al, 2010), which can adjust for gene length bias in DEGs [ 71 ]. Recombinant protein expression and cell-free assay For the expression of recombinant protein in prokaryotic cells, the CDSs of COR47 were cloned and inserted into the pMal-cRi (MBP tag) vector and transformed into the E. coli strain Rosetta, MBP tagged proteins were purified using PurKine™ MBP-Tag Dextrin Resin 6FF (BMR20206, Abbkine) following the manufacturer’s instructions. For the cell-free assay, 2000 − 400 mg of 7-day-old normally growing plants was collected, and total protein was extracted via protein degradation buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM DTT, and 5 mM ATP). Add 100 ng of purified MBP-COR47 protein, and the mixture was incubated at room temperature for 0, 15, 30, 45, or 60 minutes. Equal amounts of the reaction mixture were collected at each time point to detect MBP-COR47 expression using anti-MBP (Abmart, 15089-1-AP, 1:5000). Transient expression in Arabidopsis protoplasts and cold treatment Transient expression assays in Arabidopsis protoplasts were conducted essentially as described previously [ 72 , 73 ]. Briefly, equal amounts (100 µg) of the COR47-GFP plasmid were independently transformed into protoplasts derived from the wild-type and the atg13ab mutant. After incubation at 22°C for 16 h to allow sufficient expression of the fusion protein, the protoplasts were subjected to 4°C treatment for varying durations. Total protein was then extracted and analyzed by immunoblotting with anti-GFP and anti-Actin antibodies. Conclusions In this study, we revealed that unlike its well-described positive roles in common stresses such as salt, drought, and submergence, autophagy functions as a negative regulator of cold stress responses in Arabidopsis. The autophagy-deficient mutants exhibited enhanced freezing tolerance regardless of cold acclimation. Under prolonged chilling conditions, autophagy positively regulates anthocyanin accumulation. Cold treatment significantly suppressed autophagic activity, as evidenced by the transcriptional downregulation of autophagy-related genes, reduced autophagosome formation, and decreased ATG8 protein abundance. Notably, cold-responsive genes were constitutively upregulated in the autophagy mutants, which is consistent with the persistent accumulation of the cold-regulated protein COR47. These findings collectively demonstrate the suppressive role of autophagy in low-temperature adaptation in Arabidopsis. Abbreviations ANS anthocyanin synthase ATG AuTophaGy B1L bypass1-like CBF c-repeat binding factors CHS chalcone synthase ConA concanavalin A COR47 cold-regulated 47 DAS days after sowing DDF1 dwarf and delayed flowering 1 DEG differentially expressed gene DEP differentially expressed protein DFR dihydroflavonol 4-reductase DREB dehydration-responsive element binding GFP green fluorescent protein Gols3 galactinol synthase 3 GUS glucuronidas LTI30 low-temperature-induced 30 NBR1 neighbor of BRCA1 PE phosphatidylethanolamine PI3K Phosphatidylinositol 3-kinase PI3P phosphatidylinositol 3-phosphate RD29A responsive to desiccation 29a SNARE soluble NSF attachment protein receptor UPS ubiquitin-proteasome system ZAT12 zinc finger of Arabidopsis thaliana 12 Declarations Consent for publication Not applicable. Availability of data and materials All the data generated or analyzed during this study are included in this published article [and its supplementary information files]. Competing interests The authors declare that they have no competing interests. Funding This research was financially supported by the National Natural Science Foundation of China (32400246) and the Shaanxi Provincial Natural Science Fund (22JR5RA831). Author contributions All authors contributed to the study’s conception and design. Q.L.W designed the research, Q.L.W, Y.S.P, S.J.G, B.L carried out the experiments, Q.L.W wrote the manuscript. Acknowledgments We thank Prof. FaQiang Li (South China Agricultural University) for providing the atg1abct and atg13ab mutants. Generative AI statement The author(s) declare that no generative AI was used in the creation of this manuscript. References Ding Y, Shi Y, Yang S. Regulatory networks underlying plant responses and adaptation to cold stress. Annu Rev Genet. 2024;58(1):43-65. Kosová K, Nešporová T, Vítámvás P, Vítámvás J, Klíma M, Ovesná J, Prášil IT. How to survive mild winters: Cold acclimation, deacclimation, and reacclimation in winter wheat and barley. Plant Physiol Biochem. 2025;220:109541. Ramón A, Esteves A, Villadóniga C, Chalar C, Castro-Sowinski S. A general overview of the multifactorial adaptation to cold: Biochemical mechanisms and strategies. Braz J Microbiol. 2023;54:2259-2287. Feng Y, Li Z, Kong X, Khan A, Ullah N, Zhang X. Plant coping with cold stress: Molecular and physiological adaptive mechanisms with future perspectives. Cells. 2025;14(2):110. Sun JL, Li JY, Wang MJ, Song ZT, Liu JX. Protein quality control in plant organelles: Current progress and future perspectives. Mol Plant. 2021;14:95-114. Su T, Yang M, Wang P, Zhao Y, Ma C. Interplay between the ubiquitin proteasome system and ubiquitin-mediated autophagy in plants. Cells. 2020;9(10):2219. Pohl C, Dikic I. Cellular quality control by the ubiquitin-proteasome system and autophagy. Science. 2019;366:818-822. Zientara-Rytter K, Sirko A. To deliver or to degrade – an interplay of the ubiquitin-proteasome system, autophagy and vesicular transport in plants. FEBS J. 2016; 283(19):3534-3555. Michaeli S, Galili G, Genschik P, Fernie AR, Avin-Wittenberg T. Autophagy in plants – what's new on the menu? Trends Plant Sci. 2016;21:134-144. Marshall RS, Vierstra RD. Autophagic degradation of the 26S proteasome is mediated by the dual ATG8/ubiquitin receptor RPN10 in Arabidopsis. Mol Cell. 2015;58:1053-1066. Gross AS, Raffeiner M, Zeng Y, Üstün S, Dagdas Y. Autophagy in plant health and disease. Annu Rev Plant Biol. 2025. doi: 10.1146/annurev-arplant-060324-094912. Liu Y, Bassham DC. Autophagy: Pathways for self-eating in plant cells. Annu Rev Plant Biol. 2012;63:215-237. Yoshimoto K, Ohsumi Y. Unveiling the molecular mechanisms of plant autophagy – from autophagosomes to vacuoles in plants. Plant Cell Physiol. 2018;59:1337-1344. Lv X, Pu X, Qin G, Zhu T, Lin H. The roles of autophagy in development and stress responses in Arabidopsis thaliana . Apoptosis. 2014;19:905-921. Yano K, Suzuki T, Moriyasu Y. Constitutive autophagy in plant root cells. Autophagy. 2007;3:360-362. Bassham DC. Function and regulation of macroautophagy in plants. Biochim Biophys Acta. 2009;1793:1397-1403. Yoshimoto K, Hanaoka H, Sato S, Kato T, Tabata S, Noda T, Ohsumi Y. Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy. Plant Cell. 2004;16:2967-2983. Chung T, Phillips AR, Vierstra RD. ATG8 lipidation and ATG8‐mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci. Plant J. 2010;62:483-493. Suttangkakul A, Li F, Chung T, Vierstra RD. The ATG1/ATG13 protein kinase complex is both a regulator and a target of autophagic recycling in Arabidopsis. Plant Cell. 2011;23:3761-3779. Naumann C, Müller J, Sakhonwasee S, Wieghaus A, Hause G, Heisters M, Bürstenbinder K, Abel S. The local phosphate deficiency response activates endoplasmic reticulum stress dependent autophagy. Plant Physiol. 2019;179:460-476. Lornac A, Havé M, Chardon F, Soulay F, Clément G, Avice JC, Masclaux-Daubresse C. Autophagy controls sulphur metabolism in the rosette leaves of Arabidopsis and facilitates S remobilization to the seeds. Cells. 2020;9(2):332. Liu Y, Xiong Y, Bassham DC. Autophagy is required for tolerance of drought and salt stress in plants. Autophagy. 2009;5:954-963. Luo L, Zhang P, Zhu R, Fu J, Su J, Zheng J, Wang Z, Wang D, Gong Q. Autophagy is rapidly induced by salt stress and is required for salt tolerance in Arabidopsis. Front Plant Sci. 2017;8:1459. Bao Y, Song W-M, Wang P, Yu X, Li B, Jiang C, Shiu S-H, Zhang H, Bassham DC. COST1 regulates autophagy to control plant drought tolerance. Pro Natl Acad Sci. 2020;117(13):7482-7493. Chen L, Liao B, Qi H, Xie LJ, Huang L, Tan WJ, Zhai N, Yuan LB, Zhou Y, Yu LJ, Chen QF, Shu W, Xiao S. Autophagy contributes to regulation of the hypoxia response during submergence in Arabidopsis thaliana . Autophagy. 2015;11:2233-2246. Thirumalaikumar VP, Gorka M, Schulz K, Masclaux-Daubresse C, Sampathkumar A, Skirycz A, Vierstra RD, Balazadeh S. Selective autophagy regulates heat stress memory in Arabidopsis by NBR1-mediated targeting of HSP90.1 and ROF1. Autophagy. 2021;17:2184-2199. Sedaghatmehr M, Thirumalaikumar VP, Kamranfar I, Marmagne A, Masclaux-Daubresse C, Balazadeh S. A regulatory role of autophagy for resetting the memory of heat stress in plants. Plant Cell Environ. 2019;42:1054-1064. Zhai Y, Guo M, Wang H, Lu J, Liu J, Zhang C, Gong Z, Lu M. Autophagy, a conserved mechanism for protein degradation, responds to heat, and other abiotic stresses in Capsicum annuum L. Front Plant Sci. 2016;7:131. Chi C, Li X, Fang P, Xia X, Shi K, Zhou Y, Zhou J, Yu J. Brassinosteroids act as a positive regulator of NBR1-dependent selective autophagy in response to chilling stress in tomato. J Exp Bot. 2020;71:1092-1106. Li Q, Wang B, Yu H. New mechanism of strigolactone-regulated cold tolerance in tomato. New Phytol. 2025;245:921-923. Chen XL, Zheng XL, Xu T, Zou JP, Jin WD, Wang GH, Yang P, Zhou J. Role of selective autophagy receptors in tomato response to cold stress. Environ Exp Bot. 2023;213:105426. Zhao W, Song J, Wang M, Chen X, Du B, An Y, Zhang L, Wang D, Guo C. Alfalfa MsATG13 confers cold stress tolerance to plants by promoting autophagy. Int J Mol Sci. 2023;24(15):12033. Usadel B, Bläsing OE, Gibon Y, Poree F, Höhne M, Günter M, Trethewey R, Kamlage B, Poorter H, Stitt M. Multilevel genomic analysis of the response of transcripts, enzyme activities and metabolites in Arabidopsis rosettes to a progressive decrease of temperature in the non-freezing range. Plant Cell Environ. 2008;31:518-547. Klińska-Bąchor S, Kędzierska S, Demski K, Banaś A. Phospholipid: Diacylglycerol acyltransferase1-overexpression stimulates lipid turnover, oil production and fitness in cold-grown plants. BMC Plant Biol. 2023;23:370. Sato A, Inayoshi S, Kitawaki K, Mihara R, Yoneda K, Ito-Inaba Y, Inaba T. Autophagy is suppressed by low temperatures and is dispensable for cold acclimation in Arabidopsis. Physiol Plantarum. 2024;176:e14409. Huang X, Zheng C, Liu F, Yang C, Zheng P, Lu X, Tian J, Chung T, Otegui MS, Xiao S, Gao C, Vierstra RD, Li F. Genetic analyses of the Arabidopsis ATG1 kinase complex reveal both kinase-dependent and independent autophagic routes during fixed-carbon starvation. Plant Cell. 2019;31:2973-2995. Ghosh UK, Islam MN, Siddiqui MN, Cao X, Khan MAR. Proline, a multifaceted signalling molecule in plant responses to abiotic stress: Understanding the physiological mechanisms. Plant Biol. 2022;24(2):227-239. Schulz E, Tohge T, Zuther E, Fernie AR, Hincha DK. Natural variation in flavonol and anthocyanin metabolism during cold acclimation in Arabidopsis thaliana accessions. Plant Cell Environ. 2015;38(8):1658-1672. Zhou H, He J, Zhang Y, Zhao H, Sun X, Chen X, Liu X, Zheng Y, Lin H. RHA2b-mediated MYB30 degradation facilitates MYB75-regulated, sucrose-induced anthocyanin biosynthesis in Arabidopsis seedlings. Plant Commun. 2024;5:100744. Xie Y, Tan H, Ma Z, Huang J. DELLA proteins promote anthocyanin biosynthesis via sequestering MYBL2 and JAZ suppressors of the MYB/bHLH/WD40 complex in Arabidopsis thaliana . Mol Plant. 2016;9:711-721. Masclaux-Daubresse C, Clément G, Anne P, Routaboul J-M, Guiboileau A, Soulay F, Shirasu K, Yoshimoto K. Stitching together the multiple dimensions of autophagy using metabolomics and transcriptomics reveals impacts on metabolism, development, and plant responses to the environment in Arabidopsis. Plant Cell. 2014;26(5):1857-1877. Xiao S, Gao W, Chen QF, Chan SW, Zheng SX, Ma J, Wang M, Welti R, Chye ML. Overexpression of Arabidopsis acyl-CoA binding protein ACBP3 promotes starvation-induced and age-dependent leaf senescence. Plant Cell. 2010;22:1463-1482. Qi H, Wang Y, Bao Y, Bassham DC, Chen L, Chen QF, Hou S, Hwang I, Huang L, Lai Z, Li F, Liu Y, Qiu R, Wang H, Wang P, Xie Q, Zeng Y, Zhuang X, Gao C, Jiang L, Xiao S. Studying plant autophagy: Challenges and recommended methodologies. Adv Biotechnol. 2023;1:2. Kang HG, Kim J, Kim B, Jeong H, Choi SH, Kim EK, Lee HY, Lim PO. Overexpression of FTL1/DDF1, an AP2 transcription factor, enhances tolerance to cold, drought, and heat stresses in Arabidopsis thaliana . Plant Sci. 2011;180:634-641. Jia Y, Ding Y, Shi Y, Zhang X, Gong Z, Yang S. The cbfs triple mutants reveal the essential functions of CBFs in cold acclimation and allow the definition of CBF regulons in Arabidopsis. New Phytol. 2016;212:345-353. Chen T, Chen JH, Zhang W, Yang G, Yu LJ, Li DM, Li B, Sheng HM, Zhang H, An LZ. BYPASS1-LIKE, a DUF793 family protein, participates in freezing tolerance via the CBF pathway in Arabidopsis. Front Plant Sci. 2019;10:807. Gismondi M, Strologo L, Gabilondo J, Budde C, Drincovich MF, Bustamante C. Characterization of ZAT12 protein from Prunus persica : role in fruit chilling injury tolerance and identification of gene targets. Planta. 2024;261:14. Zhang L, Jiang X, Liu Q, Ahammed GJ, Lin R, Wang L, Shao S, Yu J, Zhou Y. The HY5 and MYB15 transcription factors positively regulate cold tolerance in tomato via the CBF pathway. Plant Cell Environ. 2020;43:2712-2726. Cho Y, Kwon H, Kim BC, Shim D, Ha J. Identification of genetic factors influencing flavonoid biosynthesis through pooled transcriptome analysis in mungbean sprouts. Front Plant Sci. 2025;16:1540674. Li S, Wang W, Gao J, Yin K, Wang R, Wang C, Petersen M, Mundy J, Qiu JL. MYB75 phosphorylation by MPK4 is required for light-induced anthocyanin accumulation in Arabidopsis. Plant Cell. 2016, 28(11):2866-2883. Li S, He L, Yang Y, Zhang Y, Han X, Hu Y, Jiang Y. INDUCER OF CBF EXPRESSION 1 promotes cold-enhanced immunity by directly activating salicylic acid signaling. Plant Cell. 2024;36(7):2587-2606. Kidokoro S, Shinozaki K, Yamaguchi-Shinozaki K. Transcriptional regulatory network of plant cold-stress responses. Trends Plant Sci. 2022;27:922-935. Cheng S, Fan S, Yang C, Hu W, Liu F. Proteomics revealed novel functions and drought tolerance of Arabidopsis thaliana protein kinase ATG1. BMC Plant Biol. 2025;23:48. Li L, Lee CP, Ding X, Qin Y, Wijerathna-Yapa A, Broda M, Otegui MS, Millar AH. Defects in autophagy lead to selective in vivo changes in turnover of cytosolic and organelle proteins in Arabidopsis. Plant Cell. 2022;34:3936-3960. Hernández-Sánchez IE, Maruri-López I, Graether SP, Jiménez-Bremont JF. In vivo evidence for homo- and heterodimeric interactions of Arabidopsis thaliana dehydrins AtCOR47, AtERD10, and AtRAB18. Sci Rep. 2017;7(1):17036. Avin-Wittenberg T. Autophagy and its role in plant abiotic stress management. Plant Cell Environ. 2019;42(3):1045-1053. Yagyu M, Yoshimoto K. New insights into plant autophagy: molecular mechanisms and roles in development and stress responses. J Exp Bot. 2024;75:1234-1251. Pourcel L, Irani NG, Lu Y, Riedl K, Schwartz S, Grotewold E. The formation of anthocyanic vacuolar inclusions in Arabidopsis thaliana and implications for the sequestration of anthocyanin pigments. Mol Plant. 2010;3(1):78-90. Yang C, Luo M, Zhuang X, Li F, Gao C. Transcriptional and epigenetic regulation of autophagy in plants. Trends Genet. 2020;36(9):676-688 Quinn PJ. Effects of temperature on cell membranes. Symp Soc Exp Biol. 1988;42:237-258. Liu H, Wang FF, Peng XJ, Huang JH, Shen SH. Global phosphoproteomic analysis reveals the defense and response mechanisms of jatropha curcas seedling under chilling stress. Int J Mol Sci. 2019;20(1):208. Li X, Cai J, Liu F, Dai T, Cao W, Jiang D. Cold priming drives the sub-cellular antioxidant systems to protect photosynthetic electron transport against subsequent low temperature stress in winter wheat. Plant Physiol Biochem. 2014;82:34-43. Wu Z, Han S, Zhou H, Tuang ZK, Wang Y, Jin Y, Shi H, Yang W. Cold stress activates disease resistance in Arabidopsis thaliana through a salicylic acid dependent pathway. Plant Cell Environ. 2019;42(9):2645-2663. van Hulten M, Pelser M, van Loon LC, Pieterse CM, Ton J: Costs and benefits of priming for defense in Arabidopsis. Proc Natl Acad Sci. 2006;103(14):5602-5607. Guo W, Ward RW, Thomashow MF. Characterization of a cold-regulated wheat gene related to Arabidopsis COR47. Plant Physiol. 1992;100:915-922. Szlachtowska Z, Rurek M. Plant dehydrins and dehydrin-like proteins: Characterization and participation in abiotic stress response. Front Plant Sci. 2023;14:1213188. Rorat T. Plant dehydrins-tissue location, structure and function. Cell Mol Biol Lett. 2006;11:536-556. Li X, Liu Q, Feng H, Deng J, Zhang R, Wen J, Dong J, Wang T. Dehydrin MtCAS31 promotes autophagic degradation under drought stress. Autophagy. 2020;16:862-877. Wang X, Zhang X, Song CP, Gong Z, Yang S, Ding Y. PUB25 and PUB26 dynamically modulate ICE1 stability via differential ubiquitination during cold stress in Arabidopsis. Plant Cell. 2023;35:3585-3603. Wang Q, Qin Q, Su M, Li N, Zhang J, Liu Y, Yan L, Hou S. Type one protein phosphatase regulates fixed-carbon starvation-induced autophagy in Arabidopsis. Plant Cell. 2022;34(11):4531-4553. Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: Accounting for selection bias. Genome Biol. 2010;11:R14. Miao Y, Jiang L. Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells. Nat Protoc. 2007;2, 2348-2353. Zhuang X, Wang H, Lam SK, Gao C, Wang X, Cai Y, Jiang L. A BAR-domain protein SH3P2, which binds to phosphatidylinositol 3-phosphate and ATG8, regulates autophagosome formation in Arabidopsis. Plant Cell. 2013;25:4596-4615. Additional Declarations No competing interests reported. Supplementary Files SupplementalData.docx SupplementalDataSet1.Col0hvsCol3h.xls SupplementalDataSet2.Col0hvsCol24h.xls SupplementalDataSet3.atg13ab0hvsatg13ab3h.xls SupplementalDataSet4.atg13ab0hvsatg13ab24h.xls SupplementalDataSet5.Col0hvsatg13ab0h.xls SupplementalDataSet6.Col3hvsatg13ab3h.xls SupplementalDataSet7.Col24hvsatg13ab24h.xls Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 30 Jul, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 14 May, 2025 Editor assigned by journal 13 May, 2025 Submission checks completed at journal 13 May, 2025 First submitted to journal 08 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6620529","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":453786681,"identity":"243ff83e-a273-4705-a592-5c8483842d08","order_by":0,"name":"Yushi Peng","email":"","orcid":"","institution":"Institute of Future Agriculture, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Yushi","middleName":"","lastName":"Peng","suffix":""},{"id":453786682,"identity":"322c9d9b-3a40-4900-a5f4-924f07a6ed85","order_by":1,"name":"Shujuan Guo","email":"","orcid":"","institution":"Institute of Future Agriculture, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Shujuan","middleName":"","lastName":"Guo","suffix":""},{"id":453786683,"identity":"62fa2fd6-f9c5-464e-870a-3b19036d9582","order_by":2,"name":"Ben Lei","email":"","orcid":"","institution":"Institute of Future Agriculture, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Ben","middleName":"","lastName":"Lei","suffix":""},{"id":453786684,"identity":"5818d0ec-b8d7-46c2-807d-f7b4bdef763f","order_by":3,"name":"Qiuling Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIie3QMUvEMBTA8ZRAXV7tesWiXyFQqBwe3lcpCNclyI0dAwd1dK3Qz+H8QgaX6Dke3CLc4pAhtzuYs8stpjcK5g8hy/slJISEQn82RvLYbWibGaSpOI2AI5Hs9CLPOjztHnCLqqRVMyaqkfNfXtWnWSo4v3h4V6DXwAhGds89RN8vpj1TEOd6KftmC9dU0Ozp+XdSIi8LOJAJr9DoLUwFxjTxkbU5Ikn7BgyrEbLhxW4gNcqkxXEy35gy6ll9uIW4T76DrJMr71uyR15Y83VzedXVO2ub23marqTde4grnsDP7r53KBLeeRe1Azn7GJsMhUKhf9o31zhXATaa5q4AAAAASUVORK5CYII=","orcid":"","institution":"Institute of Future Agriculture, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A\u0026F University","correspondingAuthor":true,"prefix":"","firstName":"Qiuling","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-05-08 12:08:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6620529/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6620529/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-07066-9","type":"published","date":"2025-07-30T16:20:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82337072,"identity":"3f07157d-6401-4d7f-a3f8-475252319f42","added_by":"auto","created_at":"2025-05-09 08:28:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":691102,"visible":true,"origin":"","legend":"\u003cp\u003eAutophagy mutants exhibit tolerance to freezing stress in Arabidopsis. \u003cstrong\u003eA \u003c/strong\u003eFreezing phenotype of the autophagy mutants under cold-acclimated (CA) and non-acclimated (NA) conditions. 14-day-old seedlings grown on half-strength MS (1/2 MS) plates at 22℃ were treated at −10℃ for 2 h after pre-treatment at 4℃ for 2 days (CA) or were treated at −5℃ for 2 h (NA).\u003cstrong\u003e B, C \u003c/strong\u003eChlorophyll contents of the seedlings shown in (A).\u003cstrong\u003e D, E\u003c/strong\u003e Ion leakage of the seedlings shown in (A). \u003cstrong\u003eF \u003c/strong\u003eProline content of Col and autophagy mutants at 22°C or 4°C. In (B-F), each bar represents the mean ± SD of three independent experiments. Different letters above the columns indicate significant differences determined using one-way ANOVA withTukey’s multiple comparison analyses (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/d3f220ea73d22e9820576885.png"},{"id":82335693,"identity":"8c041623-b798-49d4-88f4-2822f827160c","added_by":"auto","created_at":"2025-05-09 08:12:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":450199,"visible":true,"origin":"","legend":"\u003cp\u003eAutophagy regulates anthocyanin accumulation in Arabidopsis under chilling conditions. \u003cstrong\u003eA \u003c/strong\u003eWT and autophagy mutants grown at 22°C or 4°C. Five-day-old seedlings were transplanted to 1/2 MS medium containing 1% agar and grown at 22°C for an additional 8 d or 4°C for an additional 45 d before photographs were taken. \u003cstrong\u003eB \u003c/strong\u003eAnthocyanin content of the seedlings shown in (A), FW, fresh weight. \u003cstrong\u003eC \u003c/strong\u003eqRT-PCR analysis of the expression of selected anthocyanin biosynthetic genes in WT and \u003cem\u003eatg13ab\u003c/em\u003emutant upon 4°C treatment for indicated times.\u003cem\u003e Ubiquitin10 \u003c/em\u003e(\u003cem\u003eUBQ10\u003c/em\u003e) was used as an internal control. In (B, C), each bar represents the mean ± SD of three independent experiments. Different letters above the columns indicate significant differences determined using one-way ANOVA with Tukey’s multiple comparison analyses (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/7b3bcef3ea642cc6320cd35e.png"},{"id":82335695,"identity":"ceb182ec-50c3-4a35-a583-3b4cc59967fd","added_by":"auto","created_at":"2025-05-09 08:12:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":327676,"visible":true,"origin":"","legend":"\u003cp\u003eCold inhibits the expression of autophagy-related genes. \u003cstrong\u003eA\u003c/strong\u003e qRT-PCR analysis of the expression of selected autophagy-related genesin WT upon 4°C treatment for indicated times.\u003cem\u003e Ubiquitin10 \u003c/em\u003e(\u003cem\u003eUBQ10\u003c/em\u003e) was used as an internal control. Each bar represents the mean ± SD of three independent experiments. Asterisks indicate significant differences as determined by Student’s \u003cem\u003et\u003c/em\u003e-test (*** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001). \u003cstrong\u003eB \u003c/strong\u003eGUS histochemical staining of the 10-day-old transgenic lines expressing \u003cem\u003eGUS\u003c/em\u003egene driven by \u003cem\u003eATG8a\u003c/em\u003e and \u003cem\u003eATG13a\u003c/em\u003e promoter upon 4°C treatment for indicated times. Bars = 7 mm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/8bb3489a147d9db0992117fa.png"},{"id":82335698,"identity":"0039b170-d622-45aa-84fb-fcd31e425b1c","added_by":"auto","created_at":"2025-05-09 08:12:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":451913,"visible":true,"origin":"","legend":"\u003cp\u003eAutophagic flux is reduced upon cold treatment. \u003cstrong\u003eA \u003c/strong\u003eDeposition of GFP-ATG8a-labeled autophagic bodies inside the central vacuole of WT at 22°C or 4°C. seedlings of \u003cem\u003eproATG8a::GFP-ATG8a\u003c/em\u003e/Col grown on 1/2 MS agar medium for 5 days at 22°C were transferred to 1/2 MS liquid medium and either continued culturing at 22°C for 12 h (control), or exposed to 4°C for 12 h under a standard photoperiod. Seedlings were then allowed to recover for 6 h at 22°C. Subsequently, either DMSO (control) or 0.5 μM ConA was added to the liquid medium, followed by further incubation at 22°C for 4 h before microscopy observation. Concanamycin A (ConA), 0.5 μM. Scale bars, 10 µm.\u003cstrong\u003e B \u003c/strong\u003eQuantification of the number of autophagic bodies shown in (A). Four different images in the root elongation zone were photographed per seedling and the number of puncta in each image was counted and averaged. A total of ten seedlings were observed per treatment. Data represent mean ± SD (\u003cem\u003en\u003c/em\u003e= 40), analyzed by one-way ANOVA followed by Tukey’s multiple comparison analyses (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Different letters represent significantly different means. \u003cstrong\u003eC \u003c/strong\u003eImmunoblotting analysis showing the processing of GFP-ATG8a in WT upon 4°C treatment for indicated times. \u003cstrong\u003eD \u003c/strong\u003eRelative ratio of free GFP to GFP-ATG8a shown in (C), as quantified by ImageJ. \u003cstrong\u003eE \u003c/strong\u003eImmunoblot detecting the ATG8 lipidation level in Col upon 4°C treatment for indicated times. \u003cstrong\u003eF \u003c/strong\u003eRelative intensity of total ATG8 protein normalized to the loading control Actin shown in (E). In (D, F), each bar represents the mean ± SD of three independent experiments, asterisks indicate significant differences as determined by Student’s \u003cem\u003et\u003c/em\u003e-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, *** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001). All protein experiments were repeated at least 3 times with similar results. Actin was used as a protein loading control. hpt, hours post-treatment.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/b547187cc4393ad8619a71b6.png"},{"id":82335711,"identity":"e4eed5fd-ef7f-4263-ab6a-9f444a7af712","added_by":"auto","created_at":"2025-05-09 08:12:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":605797,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentially expressed gene (DEG) analysis between Col and \u003cem\u003eatg13ab\u003c/em\u003emutant. \u003cstrong\u003eA \u003c/strong\u003eHeatmap of DEGs in Col and \u003cem\u003eatg13ab\u003c/em\u003e before or after cold treatment. The genes analyzed included the overlap of DEGs in \u003cem\u003eatg13ab\u003c/em\u003eas compared with Col. DEGs were defined as FDR \u0026lt; 0.05 \u0026amp; |Log2 (foldchange)| ≥ 1. \u003cstrong\u003eB \u003c/strong\u003eNumber of up- and down-regulated DEGs between cold-treated for 3 or 24 h venus untreated (0 hour of cold treatment) samples in Col and \u003cem\u003eatg13ab\u003c/em\u003e.\u003cstrong\u003eC, D \u003c/strong\u003eThe cold induced (C) or repressed (D) genes expression in Col and \u003cem\u003eatg13ab\u003c/em\u003e. Fourteen-day-old seedlings were treated at 4°C for 0, 3, 24 h for RNA-Seq assay. Each sample was compared with 0 h time point to choose the cold induced (log2 ≥ 1, FDR ≤ 0.05) or repressed (log2 ≤ 1, FDR ≤ 0.05) genes. The differentially expressed genes were picked out to draw the Venn diagram. \u003cstrong\u003eE \u003c/strong\u003eNumber of up- and down-regulated DEGs between Col and \u003cem\u003eatg13ab\u003c/em\u003e at indicated hours of cold treatment.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/f74acda7e377b6a3a844ab0d.png"},{"id":82335713,"identity":"e9c8f95c-9718-4ce9-9d98-d173d2dbaf6c","added_by":"auto","created_at":"2025-05-09 08:12:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":592225,"visible":true,"origin":"","legend":"\u003cp\u003eGO enrichment analysis of DEGs between Col and \u003cem\u003eatg13ab\u003c/em\u003emutant at 0 hour of cold treatment. \u003cstrong\u003eA, B \u003c/strong\u003eThe enrichment analysis of up-regulated (A) and down-regulated (B) DEGs was performed using the GO term that describes biological process. Fold change ≥ 2, P‐value (FDR) \u0026lt; 0.01. \u003cstrong\u003eC \u003c/strong\u003eClustering display of the induced cold and freezing‐related DEGs in \u003cem\u003eatg13ab \u003c/em\u003ecompared to Col before cold treatment. \u003cstrong\u003eD\u003c/strong\u003e Clustering display of the repressed flavonoid biosynthetic‐related DEGs in in\u003cem\u003e atg13ab\u003c/em\u003e compared to Col before cold treatment.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/e028777f1ebda1b48c1f549d.png"},{"id":82335705,"identity":"86e70e80-f9e1-4de0-a7f4-e3918a1b2661","added_by":"auto","created_at":"2025-05-09 08:12:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":914594,"visible":true,"origin":"","legend":"\u003cp\u003eGO enrichment analysis of DEGs between Col and \u003cem\u003eatg13ab\u003c/em\u003emutant upon cold treatment at 3 h and 24 h. The enrichment analysis of up-regulated at 3 h (A), down-regulated 3 h (B), up-regulated at 24 h (C), down-regulated at 24 h (D).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/c8238e1798a5485888f2cafc.png"},{"id":82335714,"identity":"a5674d17-d49f-4af8-b71c-41766f0ef40b","added_by":"auto","created_at":"2025-05-09 08:12:11","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":259463,"visible":true,"origin":"","legend":"\u003cp\u003eVerification of the expression levels of cold-related genes in\u003cem\u003e atg13ab \u003c/em\u003ecompared to Col before and after cold treatment by qRT-PCR. \u003cem\u003eUBQ10\u003c/em\u003e was used as a reference gene. Each bar represents the mean ± SD of three independent experiments. Asterisks indicate significant differences as determined by Student’s \u003cem\u003et\u003c/em\u003e-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/35af642e0f024a81b885ab90.png"},{"id":82336084,"identity":"ba76ff13-4d69-4349-9a3f-67080e7ded2e","added_by":"auto","created_at":"2025-05-09 08:20:12","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":330217,"visible":true,"origin":"","legend":"\u003cp\u003eCOR47 accumulated in autophagy mutant \u003cem\u003eatg13ab\u003c/em\u003e.\u003cstrong\u003e A, B \u003c/strong\u003eDegradation of COR47 was inhibited in the autophagy mutant \u003cem\u003eatg13ab\u003c/em\u003e in the cell-free degradation assay. Recombinant purified MBP-COR47 was incubated in equal amounts of total proteins extracted from 7-day-old Col and \u003cem\u003eatg13ab \u003c/em\u003eseedlings in the presence of 1 mM ATP with or without 5 μM MG132. MBP-COR47 was detected with anti-MBP antibody. Relative intensity of MBP-COR47 normalized to the loading control Actin shown in (B). \u003cstrong\u003eC, D \u003c/strong\u003eThe stability of COR47-GFP in Col and\u003cem\u003e atg13ab\u003c/em\u003e at 22°C. Constructs encoding COR47-GFP were transfected into Arabidopsis protoplasts in the presence of 5 μM MG132, the COR47 protein was detected by immunoblotting with an anti-GFP antibody. \u003cstrong\u003eE, F \u003c/strong\u003eThe stability of COR47-GFP in Col and\u003cem\u003e atg13ab\u003c/em\u003e at 4°C. The red asterisk (*) indicates nonspecific bands. In (B, D, F), each bar represents the mean ± SD of three independent experiments, asterisks indicate significant differences as determined by Student’s \u003cem\u003et\u003c/em\u003e-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001). All protein experiments were repeated at least 3 times with similar results. hpt, hours post-treatment.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/8bd1945c11e6fc3f96772e95.png"},{"id":88268156,"identity":"aa2b0fd0-ed00-4212-8598-8e7477c8f792","added_by":"auto","created_at":"2025-08-04 16:49:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5800595,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/e8618477-ef0f-4bf8-be40-b61158ee0bae.pdf"},{"id":82335691,"identity":"75b0e185-ba0b-4574-823b-9c527b063774","added_by":"auto","created_at":"2025-05-09 08:12:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14755,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalData.docx","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/fc58b742b7c8d0a210f80876.docx"},{"id":82337417,"identity":"cd00139f-e31c-4d73-bc72-24710dfb2fa0","added_by":"auto","created_at":"2025-05-09 08:36:11","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1011523,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalDataSet1.Col0hvsCol3h.xls","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/a77fb1fabcb27bb8d8e8798d.xls"},{"id":82335706,"identity":"b0cc26eb-30e3-405f-9f47-f4c1788e931b","added_by":"auto","created_at":"2025-05-09 08:12:11","extension":"xls","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2862594,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalDataSet2.Col0hvsCol24h.xls","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/b2f6d08b51c03d6998997a06.xls"},{"id":82336080,"identity":"3b5bb4d6-df60-402c-a4b9-200cccd03895","added_by":"auto","created_at":"2025-05-09 08:20:11","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1229672,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalDataSet3.atg13ab0hvsatg13ab3h.xls","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/c05b56c2efdc76dc688f29d0.xls"},{"id":82335712,"identity":"c4a2eef1-2512-40b3-893b-661cf9225e6b","added_by":"auto","created_at":"2025-05-09 08:12:11","extension":"xls","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3208188,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalDataSet4.atg13ab0hvsatg13ab24h.xls","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/f59179c69af943079a6d78aa.xls"},{"id":82337074,"identity":"f2041d89-df95-4087-a34f-99fb8f21d17d","added_by":"auto","created_at":"2025-05-09 08:28:11","extension":"xls","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":332465,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalDataSet5.Col0hvsatg13ab0h.xls","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/04e2baea1ebbec38ccfcebea.xls"},{"id":82335720,"identity":"d3965d82-58c3-4e55-8a92-2868af69f5db","added_by":"auto","created_at":"2025-05-09 08:12:12","extension":"xls","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":172499,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalDataSet6.Col3hvsatg13ab3h.xls","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/77f2fd48ac0949b793a2fae2.xls"},{"id":82335718,"identity":"86b377e1-b28b-4e9f-9720-1fd618046eac","added_by":"auto","created_at":"2025-05-09 08:12:12","extension":"xls","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":117661,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalDataSet7.Col24hvsatg13ab24h.xls","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/7883d51a20f7912ce20e455d.xls"},{"id":82335715,"identity":"5b91dd1b-3f5a-4d9b-bcfd-3e664c78a99f","added_by":"auto","created_at":"2025-05-09 08:12:11","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":2474217,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6620529/v1/090eec04643b67e570241c6f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eInhibition of Autophagy Increases Freezing Survival in \u003cem\u003eArabidopsis\u003c/em\u003e \u003cem\u003ethaliana\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlants face dual challenges of biotic and abiotic stresses at different growth stages. Cold stress, a critical environmental factor limiting plant geographical distribution and agricultural production, significantly inhibits plant growth and development by disrupting cell membrane integrity, inducing oxidative stress and interfering with metabolic homeostasis [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Cold stress triggers the abnormal accumulation of denatured substances (such as misfolded proteins and damaged organelles) in plant cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. If these abnormal components are not promptly cleared, they form toxic aggregates, disrupt cellular metabolism, inhibit growth and development, and weaken stress resistance [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To address the clearance of damaged proteins under stress conditions, plants have evolved two classical degradation pathways: the autophagy-dependent degradation pathway and the ubiquitin-dependent 26S proteasome degradation pathway [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The ubiquitin‒proteasome system (UPS) primarily degrades soluble small-molecule proteins and peptides tagged with polyubiquitin chains, while autophagy is responsible for clearing insoluble protein aggregates, large protein complexes, intracellular pathogens, and damaged organelles [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The synergistic action of these two pathways forms the core framework of the plant protein quality control network.\u003c/p\u003e \u003cp\u003eThe autophagy process involves a membrane-independent extension structure that can encapsulate cytoplasmic components [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This process relies on the coordinated action of various ATG (AuTophaGy-related) proteins. In plants, at least 36 ATG proteins have been identified, forming different complexes that regulate various stages of autophagy: initiation, vesicle nucleation, elongation, maturation, fusion of autophagosomes with vacuoles, and degradation of autophagosomes [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Among these, the ATG1 kinase complex (ATG1-ATG13-ATG11-ATG101) is responsible for sensing the nutritional status of cells and initiating autophagy when needed. The PI3K (phosphatidylinositol 3-kinase) complex (VPS34-VPS15-ATG6-ATG14) catalyzes the production of specific lipid signals, PI3P (phosphatidylinositol 3-phosphate), aiding in the remodeling of autophagosome membrane structures. The ATG9 complex (ATG9-ATG2-ATG18) primarily mediates lipid transport, supporting the expansion of autophagic vesicles. The two ubiquitin-like systems, ATG8-PE (phosphatidylethanolamine) and ATG5-ATG12, participate in the maturation of autophagosomes, preparing them for sealing. The SNARE (soluble NSF attachment protein receptor) complex regulates the membrane fusion of autophagosomes with vacuoles, ensuring the efficient degradation of substrates. These systems work in concert to complete the entire process of autophagy, from induction to degradation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Autophagy normally maintains basal levels of activity in plants [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] but is induced under nutrient-deficient conditions, including nitrogen, carbon, phosphorus, and sulfur [\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Additionally, autophagy also serves as a critical mechanism for plants to cope with abiotic stresses, with the majority of autophagy mutants showing increased sensitivity to salt stress [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], drought stress [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], hypoxia stress [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and heat stress [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], highlighting the role of autophagy in adaptation to abiotic stresses.\u003c/p\u003e \u003cp\u003eCurrent evidence regarding the role of plant autophagy in the cold response remains contradictory. A previous study revealed that the accumulation of autophagosomes increases and that the expression of most \u003cem\u003eCaATG\u003c/em\u003e genes is induced in \u003cem\u003eCapsicum annuum\u003c/em\u003e upon cold treatment [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], indicating that autophagy may positively regulate the plant cold stress response. Moreover, chilling stress significantly induces the transcription of a series of autophagy-related genes, such as \u003cem\u003eATG2\u003c/em\u003e, \u003cem\u003eATG6\u003c/em\u003e, and \u003cem\u003eATG8\u003c/em\u003e, and enhances the autophagic flux in tomato. Increased autophagic activity thus improves the cold tolerance of plants [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Further research revealed that among this series of cold-induced tomato \u003cem\u003eATG\u003c/em\u003e genes, \u003cem\u003eATG18a\u003c/em\u003e is also included. The mutant of \u003cem\u003eATG18a\u003c/em\u003e exhibited sensitivity to cold treatment, along with decreased autophagosome formation and increased accumulation of ubiquitinated proteins [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Cold also induces the expression of selective autophagy receptors such as \u003cem\u003eNBR1a\u003c/em\u003e (a neighbor of BRCA1), \u003cem\u003eNBR1b\u003c/em\u003e, and \u003cem\u003eSEC62\u003c/em\u003e in tomato, and silencing these selective autophagy receptors affects the generation of autophagy, leading to compromised cold tolerance [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Additionally, \u003cem\u003eATG13\u003c/em\u003e is upregulated in response to cold stress in \u003cem\u003eMedicago sativa\u003c/em\u003e, and compared with wild-type plants, transgenic tobacco overexpressing \u003cem\u003eMsATG13\u003c/em\u003e enhanced cold tolerance compared to wild-type plants due to the upregulation of other \u003cem\u003eATGs\u003c/em\u003e that are necessary for autophagosome production under cold stress conditions [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], suggesting the positive role of autophagy in plant resistance to cold stress. However, some studies indicate that autophagy in plants is restricted under low-temperature conditions. Compared with that in normal plants, the transcription of autophagy-related genes in cold-treated Arabidopsis was significantly suppressed. The levels of the autophagy marker protein ATG8 decreased substantially, whereas the protein accumulation of the autophagy substrate NBR1 increased markedly [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These observations suggest that autophagy is inhibited or impaired under cold stress. A recent study revealed that autophagy is dispensable for cold acclimation and freezing tolerance in Arabidopsis, \u003cem\u003eatg5-1\u003c/em\u003e and \u003cem\u003eatg10-1\u003c/em\u003e mutant exhibited normal freezing tolerance, cold-regulated gene expression showed no significant differences between the \u003cem\u003eatg\u003c/em\u003e mutants and wild- type as well as autophagy was rarely induced by cold exposure [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These conflicting results suggest the complex role of autophagy in plant cold stress responses, as well as the functional divergence of autophagy across species under stress conditions.\u003c/p\u003e \u003cp\u003eIn this study, we found that autophagy mutants of Arabidopsis exhibited enhanced tolerance to freezing. Autophagy was significantly suppressed by cold, both at the transcriptional and protein levels. Additionally, cold-responsive genes were constitutively activated in the autophagy mutants, and the cold-regulated protein COR47 was more stable in the autophagy defect mutants. These findings reveal that autophagy serves as a negative regulatory pathway for plant adaptation to cold stress.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAutophagy negatively regulates freezing tolerance in Arabidopsis\u003c/h2\u003e \u003cp\u003eTo explore the contributions of autophagy to freezing tolerance in Arabidopsis, various autophagy mutants (\u003cem\u003eatg1abct\u003c/em\u003e, \u003cem\u003eatg13ab\u003c/em\u003e, \u003cem\u003eatg5-1\u003c/em\u003e, and \u003cem\u003eatg7-3\u003c/em\u003e) were collected as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Fourteen-day-old seedlings were subjected to freezing treatment. Under cold-acclimated (CA) conditions, these autophagy mutants exhibited stronger freezing tolerance compared to the wild type (reflected by higher chlorophyll content) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). Next, we examined ion leakage, which is an indicator of stress-induced plasma membrane damage, in the autophagy mutants. Ion leakage in the mutants under CA conditions was consistently lower than that in the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). Since autophagy is implicated in plant stress memory, autophagy mutants that undergo heat priming retain thermotolerance memory and exhibit increased resistance to subsequent heat stress [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. We therefore investigated whether non-acclimated (NA) autophagy mutants maintained improved freezing tolerance. Consistent with their performance under CA conditions, these autophagy mutants still displayed higher chlorophyll contents and lower ion leakage rates without cold acclimation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-E). Furthermore, proline, a well-documented stress-protective metabolite in plants [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], was found to accumulate at higher levels in autophagy mutants than in wild-type plants following cold treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These findings collectively demonstrate that autophagy serves as a negative regulator of freezing tolerance in Arabidopsis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAutophagy regulates the accumulation of anthocyanins in Arabidopsis under chilling conditions\u003c/h3\u003e\n\u003cp\u003eTo validate the function of autophagy under chilling conditions in plants, the wild type and all the autophagy mutants were grown at 22\u0026deg;C and 4\u0026deg;C under normal photoperiod conditions (16 h/light and 8 h/dark), respectively. No significant differences were detected among the plants grown at 22\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, under continuous growth at 4\u0026deg;C for 45 days, the wild-type plants exhibited a noticeable accumulation of anthocyanins in their cotyledons, which was consistent with the findings of a previous study [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, the cotyledons of the mutants still maintained a distinct green color (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). As \u003cem\u003eDFR\u003c/em\u003e (dihydroflavonol 4-reductase), \u003cem\u003eCHS\u003c/em\u003e (chalcone synthase), and \u003cem\u003eANS\u003c/em\u003e (anthocyanin synthase) are key enzymes in anthocyanin biosynthesis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], further analysis of the expression of these genes in different plants revealed that the expression of \u003cem\u003eDFR\u003c/em\u003e, \u003cem\u003eCHS\u003c/em\u003e, and \u003cem\u003eANS\u003c/em\u003e was significantly suppressed in the autophagy mutants \u003cem\u003eatg13ab\u003c/em\u003e and \u003cem\u003eatg5-1\u003c/em\u003e both before and after cold treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This finding is consistent with a previous study showing down-regulation of the flavonoid biosynthetic pathway in autophagy mutants [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. These results indicate that autophagy negatively regulates anthocyanin accumulation in Arabidopsis under chilling conditions and that anthocyanins likely do not directly contribute to the increased cold tolerance observed in autophagy mutants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCold inhibits the expression of autophagy-related genes\u003c/h3\u003e\n\u003cp\u003eTo further investigate how cold affects autophagy in Arabidopsis, the transcription of autophagy-related genes were first examined after treatment at 4\u0026deg;C under normal photoperiod conditions. It was found that cold treatment significantly suppressed the expression of a series of autophagy-related genes, such as \u003cem\u003eATG8a\u003c/em\u003e, \u003cem\u003eATG8e\u003c/em\u003e, \u003cem\u003eATG1a\u003c/em\u003e, \u003cem\u003eATG13a\u003c/em\u003e, \u003cem\u003eATG5\u003c/em\u003e, and \u003cem\u003eATG7\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), which is consistent with previous reports [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. To further characterize the transcriptional regulation of autophagy-related genes, we generated stable transgenic lines by fusing the 2000-bp promoter regions upstream of the \u003cem\u003eATG8a\u003c/em\u003e and \u003cem\u003eATG13a\u003c/em\u003e transcription start sites to the β-glucuronidase (GUS) reporter gene. Histochemical GUS staining revealed that under normal growth conditions, both the \u003cem\u003eATG8a\u003c/em\u003e and \u003cem\u003eATG13a\u003c/em\u003e promoters drove strong reporter gene expression in the cotyledons and roots of Arabidopsis seedlings. However, following cold treatment at 4\u0026deg;C, we observed a significant reduction in GUS expression for both genes after 6 hours of cold exposure, and this suppression was particularly pronounced in cotyledons. The inhibitory effect became more evident after 24 hours of cold treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These results indicate that low temperatures can inhibit the transcription of autophagy genes in Arabidopsis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAutophagic flux is reduced upon cold treatment\u003c/h3\u003e\n\u003cp\u003eThe suppression of autophagy gene expression by cold treatment suggests that autophagic activity may also be inhibited after cold treatment. To validate this hypothesis, the \u003cem\u003epATG8a:GFP-ATG8a\u003c/em\u003e/Col transgenic line, a well-characterized autophagosome marker line [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], was used. In the presence of concanamycin A (ConA), an autophagy inhibitor that stabilizes autophagic bodies [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], it was observed that the number of autophagosomes labeled with GFP-ATG8a significantly decreased after cold treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). We then used the release of free GFP from GFP-ATG8a to monitor autophagic transport, as previously reported [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Consistent with the microscopy results, free GFP levels were substantially reduced upon cold treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). The lipidation of ATG8 and the cleavage efficiency of the GFP-ATG8 fusion protein are commonly used indicators of autophagic flux [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Consistent with previous findings, both lipidated and non-lipidated ATG8 rapidly decreased following cold treatment [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F). These results confirm that cold inhibits autophagic flux in Arabidopsis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferentially expressed gene (DEG) analysis between Col and the\u003c/b\u003e \u003cb\u003eatg13ab\u003c/b\u003e \u003cb\u003emutant\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further investigate the effect of autophagy on the transcriptome profile, we performed RNA-seq analysis of 14-day-old wild-type and \u003cem\u003eatg13ab\u003c/em\u003e seedlings treated at 4\u0026deg;C for 0, 3, or 24 h. Two independent experiments were carried out (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), and the differentially expressed genes were analyzed via HTSeq and DESeq2 software. At 3 h after cold treatment, 959 genes were up-regulated (log2\u0026thinsp;\u0026ge;\u0026thinsp;1, FDR\u0026thinsp;\u0026le;\u0026thinsp;0.01), and 623 genes were down-regulated (log2\u0026thinsp;\u0026le;\u0026thinsp;1, FDR\u0026thinsp;\u0026le;\u0026thinsp;0.01) in the wild-type. When cold treatment was extended to 24 h, 2163 genes were up-regulated and 2086 genes were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In the \u003cem\u003eatg13ab\u003c/em\u003e mutant, the up-regulated genes numbered 870 and 2296, respectively, at 3 h or 24 h of cold treatment, and the down-regulated genes numbered 1090 and 2367, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Moreover, a total of 1999 cold-induced genes and 1706 cold-repressed genes were identified in both the wild-type and \u003cem\u003eatg13ab\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results suggest that a large number of cold regulate genes are not affected in the \u003cem\u003eatg13ab\u003c/em\u003e mutant under cold stress.\u003c/p\u003e \u003cp\u003eFor both Col and \u003cem\u003eatg13ab\u003c/em\u003e, the number of DEGs induced at 24 h was significantly greater than that induced at 3 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), indicating that cold treatment for 24 h caused substantial transcriptional rearrangement within the plant. Additionally, it was observed that a large number of DEGs were found in both wild-type and \u003cem\u003eatg13ab\u003c/em\u003e even under normal growth conditions, while the number of DEGs in Col and \u003cem\u003eatg13ab\u003c/em\u003e decreased as the duration of cold treatment extended (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Compared with Col, untreated \u003cem\u003eatg13ab\u003c/em\u003e had 525 DEGs, with 337 genes up-regulated and 148 genes down-regulated; at 3 h of cold treatment, there were a total of 272 DEGs in Col and \u003cem\u003eatg13ab\u003c/em\u003e, with 181 upregulated and 91 downregulated; and at 24 h of cold treatment, these DEGs numbered only 161, including 37 up-regulated and 124 down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). These results indicated that cold stress greatly reduced the difference in gene expression profiles between Col and the \u003cem\u003eatg13ab\u003c/em\u003e mutant, suggesting that autophagy deficiency also has a substantial effect on gene expression but that this effect occurs primarily in untreated plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGO analysis of DEGs between unstressed Col and the\u003c/b\u003e \u003cb\u003eatg13ab\u003c/b\u003e \u003cb\u003emutant\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe wild-type and \u003cem\u003eatg13ab\u003c/em\u003e mutant displayed a large difference in gene expression profiles prior to cold treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). To understand the effects of autophagy on plant gene expression under normal temperature, we first performed GO enrichment analysis to assign biological processes to the identified DEGs between Col and \u003cem\u003eatg13ab\u003c/em\u003e at 0 h of cold treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Among the up-regulated DEGs in the \u003cem\u003eatg13ab\u003c/em\u003e mutant plants, those involved in transcription and DNA replication, the stress response, the organonitrogen compound and acid chemical response, and the osmotic stress response were substantially enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). These findings suggest that autophagy deficiency may lead to dysregulation of the cell cycle and that intracellular nitrogen metabolism, the cytoplasmic pH, and osmotic pressure may be altered. Surprisingly, we found that genes responsive to cold and freezing were also enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), including early cold-responsive genes such as the CBF (C-repeat binding factor)/DERB1 (dehydration-responsive element binding) transcription factors \u003cem\u003eCBF1\u003c/em\u003e, \u003cem\u003eCBF2\u003c/em\u003e, and \u003cem\u003eDDF1\u003c/em\u003e (dwarf and delayed flowering 1) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], along with well-characterized cold-inducible genes such as \u003cem\u003eB1L1\u003c/em\u003e (BYPASS1-LIKE), \u003cem\u003eZAT12\u003c/em\u003e (ZINC FINGER OF ARABIDOPSIS THALIANA12), and \u003cem\u003eMYB15\u003c/em\u003e [\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), which is consistent with previously reported constitutive upregulation of the cold-responsive genes \u003cem\u003eCBF1\u003c/em\u003e, \u003cem\u003eCBF2\u003c/em\u003e, \u003cem\u003eMYB15\u003c/em\u003e, and \u003cem\u003eZAT12\u003c/em\u003e at 60 DAS (days after sowing), which normally occurs in the \u003cem\u003eatg5\u003c/em\u003e mutant compared with the wild type [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], indicating that the cold signaling pathway is constitutively activated in the \u003cem\u003eatg13ab\u003c/em\u003e mutant under basal conditions. This may be the primary reason for the tolerance of the autophagy mutant to cold stress.\u003c/p\u003e \u003cp\u003eFurther analysis of the down-regulated DEGs in Col and \u003cem\u003eatg13ab\u003c/em\u003e under unstressed conditions revealed that these genes were enriched primarily in pathways related to single-organism metabolism, cellular protein modification, hydrogen peroxide metabolism, cellular detoxification, and drug transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). This suggests that the mutant has altered basal metabolism, disrupted protein homeostasis, impaired oxidative stress management, and reduced detoxification capacity and xenobiotic resistance. We also observed that genes involved in flavonoid biosynthesis were downregulated in the mutant, including the aforementioned anthocyanin biosynthesis-related genes \u003cem\u003eANS\u003c/em\u003e and \u003cem\u003eDFR\u003c/em\u003e, as well as the key anthocyanin regulatory transcription factors \u003cem\u003eMYB75\u003c/em\u003e and \u003cem\u003eNAC042\u003c/em\u003e [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These findings are consistent with our qRT-PCR results from previous chilling experiments and earlier reports demonstrating the constitutive downregulation of anthocyanin-related genes in the autophagy mutant \u003cem\u003eatg5\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Additionally, the plant defense response and incompatible interaction-related genes were down-regulated in the mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), suggesting that the mutant may have defects in plant immunity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGO analysis of DEGs between Col and the\u003c/b\u003e \u003cb\u003eatg13ab\u003c/b\u003e \u003cb\u003emutant in response to cold stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs described previously, cold triggers extensive transcriptional reprogramming in Arabidopsis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). To systematically investigate how cold affects plant gene expression, we performed GO enrichment analysis of DEGs in Col plants under cold treatment. Our analysis revealed that after 3 h and 24 h of cold exposure, the up-regulated DEGs were enriched predominantly in pathways related to transcription; DNA replication; organonitrogen compound, acid chemical, and metal ion responses; cold and osmotic stress response; and RNA processing, or metabolism (Supplementary Fig.\u0026nbsp;1A). Conversely, the down-regulated DEGs were enriched primarily in pathways such as transcription, DNA replication, single-organism metabolism, protein modification, hormone responses, and lipid/fatty acid metabolism (Supplementary Fig.\u0026nbsp;1B). Notably, we observed cold-induced up-regulation of salicylic acid-related and fungal defense-related genes in Col (Supplementary Fig.\u0026nbsp;1A), which is consistent with previous reports showing that cold activates immunity-related pathways [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe further analyzed the DEGs between Col and \u003cem\u003eatg13ab\u003c/em\u003e mutants after cold treatment. The up-regulated genes were enriched mainly in processes related to the stress response; transcription and DNA replication; acid, chemical, hexose, and disaccharide responses; and lipid or protein localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). These suggest that autophagy defects may lead to increased cellular stress, thereby activating stress responses and DNA repair mechanisms. Additionally, altered sugar metabolism could provide energy to sustain cell survival under cold conditions, whereas changes in lipid localization might help maintain membrane fluidity and prevent cold-induced membrane damage. Furthermore, a small number of cold-responsive genes remained up-regulated in the mutant even after 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), which may represent potential mechanisms for cold stress tolerance in the \u003cem\u003eatg13ab\u003c/em\u003e mutant. Surprisingly, although a small number of immune pathway-related genes were already down-regulated in \u003cem\u003eatg13ab\u003c/em\u003e under untreated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), a large proportion of down-regulated DEGs after 3 h and 24 h of cold treatment clustered in bacterial and fungal defense response pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D). These indicate that the \u003cem\u003eatg13ab\u003c/em\u003e mutant may suffer from immune deficiency, which compromises its ability to respond to pathogen threats during cold stress.\u003c/p\u003e \u003cp\u003eTo confirm the RNA-seq data, cold-associated genes, including \u003cem\u003eCBF1/2/3\u003c/em\u003e, \u003cem\u003eCOR15a\u003c/em\u003e (COLD-REGULATED 15a), \u003cem\u003eCOR47\u003c/em\u003e, \u003cem\u003eGols3\u003c/em\u003e (galactinol synthase 3), \u003cem\u003eDDF1\u003c/em\u003e, \u003cem\u003eB1L\u003c/em\u003e, and \u003cem\u003eZAT12\u003c/em\u003e, were selected for qRT-PCR [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The expression of the \u003cem\u003eCBF\u003c/em\u003e genes \u003cem\u003eDDF1\u003c/em\u003e, \u003cem\u003eB1L\u003c/em\u003e, and \u003cem\u003eZAT12\u003c/em\u003e was elevated in the mutant under unstressed conditions, which was consistent with the RNA-seq results (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). After 3 h of cold stress, \u003cem\u003eCBFs\u003c/em\u003e, \u003cem\u003eDDF1\u003c/em\u003e, \u003cem\u003eB1L\u003c/em\u003e, and \u003cem\u003eZAT12\u003c/em\u003e in \u003cem\u003eatg13ab\u003c/em\u003e still maintained high expression levels\u0026zwnj;. Although the basal expression of late cold-responsive genes (\u003cem\u003eCOR15a\u003c/em\u003e, \u003cem\u003eCOR47\u003c/em\u003e, \u003cem\u003eGlos3\u003c/em\u003e) was comparable to that of the wild type under normal conditions, their induction in the mutants was significantly greater than that in the wild type after 24 hours of cold treatment\u0026zwnj; (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These results reveal that autophagy deficiency leads to persistent activation of the cold signaling pathway in plants\u0026zwnj;.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCOR47 accumulated in the autophagy mutant\u003c/b\u003e \u003cb\u003eatg13ab\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGiven the marked up-regulation of cold-responsive genes in the mutants, we hypothesized that the proteins encoded by these genes might also accumulate at relatively high levels in these lines. Previous proteomic analyses of Col and the autophagy mutants \u003cem\u003eatg1abct\u003c/em\u003e, \u003cem\u003eatg11\u003c/em\u003e, and \u003cem\u003eatg5\u003c/em\u003e revealed elevated COR47 expression in the mutant background [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. To further investigate the stability of COR47 in the \u003cem\u003eatg13ab\u003c/em\u003e mutant, MBP-COR47 was purified in vitro, and its stability was examined with cell-free analysis. The results revealed that the degradation rate of MBP-COR47 was obviously inhibited in the mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA, B). Furthermore, the COR47-GFP fusion protein was transiently expressed in the protoplasts of Col and the \u003cem\u003eatg13ab\u003c/em\u003e mutant. At 22\u0026deg;C, the COR47 protein was expressed at low levels in the wild-type plants but was significantly elevated in the \u003cem\u003eatg13ab\u003c/em\u003e mutant plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC, D). With prolonged cold treatment, rapid induction of COR47 expression was observed in Col, while the \u003cem\u003eatg13ab\u003c/em\u003e mutant exhibited even greater accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE, F). These results suggest the constitutive accumulation of COR47 in the autophagy-deficient \u003cem\u003eatg13ab\u003c/em\u003e mutant, suggesting that COR47 may be a potential substrate of autophagy. To further investigate the relationship between COR47 and autophagy, we first analyzed its subcellular localization and found that COR47 primarily localizes to the cytoplasm rather than the nucleus (Supplementary Fig.\u0026nbsp;2), which is consistent with the findings of a previous study [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. However, neither yeast two-hybrid nor BiFC assays detected any interaction between COR47 and core autophagy-related proteins, although COR47 has been shown to form homodimers [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] (Supplementary Fig.\u0026nbsp;3). Therefore, COR47 likely regulates its stability through mechanisms independent of direct interactions with core autophagy proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAutophagy, as a protective strategy, has been demonstrated to play a positive role in plant responses to multiple stresses [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. However, in this study, we revealed a previously unrecognized negative regulatory role of autophagy in Arabidopsis freezing tolerance independent of cold acclimation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To elucidate the mechanism underlying the enhanced cold tolerance of the autophagy-deficient plants, we analyzed protective metabolites, including proline and anthocyanins. Notably, compared with wild-type plants, autophagy mutants accumulated higher levels of proline after cold treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), whereas anthocyanin accumulation was significantly lower in these mutants. These findings suggest that anthocyanins likely do not directly contribute to the cold-tolerant phenotype observed in autophagy mutants. Unlike previous reports attributing reduced anthocyanin levels in autophagy mutants to impaired vacuolar trafficking [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], our transcriptomic and qRT-PCR data demonstrated that anthocyanin biosynthesis-related genes (e.g., \u003cem\u003eDFR\u003c/em\u003e, \u003cem\u003eANS\u003c/em\u003e, \u003cem\u003eCHS\u003c/em\u003e) were constitutively downregulated in the \u003cem\u003eatg13ab\u003c/em\u003e and \u003cem\u003eatg5-1\u003c/em\u003e mutants under basal conditions, which is consistent with previous observations [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. These results imply that autophagy may suppress anthocyanin production by directly inhibiting the flavonoid biosynthetic pathway rather than affecting vacuolar transport. Nevertheless, the molecular mechanisms by which autophagy downregulates flavonoid metabolism remain unclear and warrant further investigation.\u003c/p\u003e \u003cp\u003eTo better understand the mechanistic contributions of autophagy to cold stress responses in Arabidopsis, we examined the effects of cold stress on autophagy and observed significant suppression of autophagic activity, which is consistent with previous studies [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This inhibition may stem from plants redirecting energy resources under cold stress: rather than fueling the highly energy-demanding catabolic process of autophagy, limited ATP is prioritized for the rapid synthesis of cold-adaptive enzymes, metabolites, and cryoprotective compounds (e.g., proline, soluble sugars), as well as activation of the CBF/DREB signaling pathway. However, the precise molecular mechanisms underlying cold-induced autophagy suppression remain unclear and represent an important direction for future research. Several potential mechanisms warrant consideration: (1) Cold stress might induce repressive chromatin states (e.g., H3K27me3 deposition) at autophagy gene promoters through vernalization-like epigenetic mechanisms, as autophagic genes are known to undergo dynamic epigenetic modifications [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]; (2) reduced lipid unsaturation at low temperatures could impair membrane fluidity [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], potentially disrupting interactions between autophagic precursors (e.g., phagophores) and PI3P-enriched domains, thereby hindering autophagosome assembly; and (3) in \u003cem\u003eJatropha curcas\u003c/em\u003e seedlings, cold treatment markedly increased the phosphorylation levels of ATG13 [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], suggesting that unidentified cold-activated kinases may phosphorylate autophagic components, altering their stability or function to suppress autophagic flux.\u003c/p\u003e \u003cp\u003eTo elucidate the broad role of autophagy in plant cold tolerance, we conducted comparative transcriptome analysis of wild-type Arabidopsis and autophagy-deficient \u003cem\u003eatg13ab\u003c/em\u003e mutants. We observed that even under nonstress conditions, Col and \u003cem\u003eatg13ab\u003c/em\u003e already exhibited substantial differences in gene expression, including the expression of cold-responsive genes such as \u003cem\u003eCBFs\u003c/em\u003e, \u003cem\u003eDDF1\u003c/em\u003e, \u003cem\u003eB1L\u003c/em\u003e, and \u003cem\u003eZAT12\u003c/em\u003e, which is consistent with previously reported constitutive upregulation of the cold-responsive gene \u003cem\u003eatg5\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This may suggest that autophagy deficiency places cells in a persistent state of stress, leading to constitutive activation of the CBF/DREB1 signaling pathway even in the absence of external cold stress. The pre-activation of CBF signaling in autophagy mutants mimics the \u0026ldquo;cold priming\u0026rdquo; phenomenon [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], allowing rapid deployment of cryoprotective mechanisms without prior cold exposure. However, the precise mechanism by which these genes are activated in the mutants remains unclear. One possible explanation is that certain positive regulators of the cold response, such as transcription factors (e.g., ICE1) or kinases (e.g., OST1), may serve as autophagy substrates. The accumulation of these genes in autophagy-deficient mutants could indirectly increase the transcriptional activity of CBF and related genes. Notably, in \u003cem\u003eatg13ab\u003c/em\u003e, genes associated with immunity against bacteria, fungi, and oomycetes were downregulated before and after cold treatment. Extensive studies have demonstrated that cold stress triggers a defense-like response similar to pathogen invasion, which typically leads to elevated salicylic acid (SA) levels and the activation of defense-related genes to prepare plants for sensitization to future pathogen infection [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. However, immune responses are energetically costly and may constrain plant growth under cold conditions [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The observed attenuation of cold-induced immune signaling in \u003cem\u003eatg13ab\u003c/em\u003e indicates that ATG13 may prevent overactivation of these pathways under non-pathogenic conditions to avoid autoimmunity, thereby increasing the freezing tolerance of the mutant.\u003c/p\u003e \u003cp\u003eDehydrins play crucial roles in plant responses to water deficit and are considered to contribute to the protection of fragile organellar structures under adverse conditions [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Although drought and freezing are distinct stresses, freezing-induced cellular dehydration triggers substantial accumulation of dehydrins [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Through cell-free and protoplast expression assays, we found that the COR47 protein was strongly induced by cold in wild-type plants, whereas it exhibited abnormal accumulation in autophagy mutants even under normal temperatures. This finding further explains the enhanced freezing tolerance phenotype of the autophagy-deficient mutants. A comparative proteomic analysis of wild-type and \u003cem\u003eatg1abct\u003c/em\u003e mutants under drought stress revealed significant enrichment of cold-regulated differentially expressed proteins (DEPs), including COR47, RESPONSIVE TO DESICCATION 29A (RD29A), and LOW TEMPERATURE-INDUCED 30 (LTI30), in the \u003cem\u003eatg1abct\u003c/em\u003e mutant prior to drought treatment [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Furthermore, studies in \u003cem\u003eatg11\u003c/em\u003e and \u003cem\u003eatg5\u003c/em\u003e mutants demonstrated that COR47 displayed higher protein abundance and lower degradation rates than did the wild type [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. These observations suggest a potential association between the dehydrin COR47 and autophagy, as previously described [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. However, we did not detect any direct interaction between COR47 and core autophagy proteins. This finding implies the possible existence of a specific autophagy receptor that targets COR47 as a cargo for degradation via the autophagy pathway.\u003c/p\u003e \u003cp\u003eAlthough we have uncovered that autophagy negatively regulates freezing stress tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e, this finding contrasts with the well-established protective role of autophagy in cold resistance among Solanaceae crops such as tomato and pepper [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], as silencing core ATG genes in tomato compromises chilling resistance by impairing the clearance of ubiquitinated protein aggregates [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The mechanistic divergence in autophagy-mediated temperature adaptation across species remains unresolved, potentially reflecting evolutionary divergence in climate adaptation strategies between cold-adapted plants (e.g., Arabidopsis) and temperate crops (e.g., tomato and pepper). Future investigations encompassing a broader phylogenetic spectrum of plant species will be critical for revealing the functional diversity of autophagy in plant cold adaptation.\u003c/p\u003e \u003cp\u003eIn summary, our study demonstrated that autophagy functions as a negative regulator in Arabidopsis in response to cold stress. This contrasts with its well-documented protective roles in other stress conditions, highlighting autophagy as a \u0026ldquo;double-edged sword\u0026rdquo; in plant stress adaptation. The complex biological outcomes of autophagy, whether promoting or suppressing cell death under stress, depend on intricate contextual factors that warrant further investigation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e \u003cp\u003eAll materials used in this work were in the \u003cem\u003eArabidopsis thaliana\u003c/em\u003e accession Columbia (Col-0) background. Mutants of \u003cem\u003eatg5-1\u003c/em\u003e (SAIL_129_B07) and \u003cem\u003eatg7-3\u003c/em\u003e (SAIL_11_H07) were obtained from the Arabidopsis Biological Resource Center (ABRC). \u003cem\u003eatg13ab\u003c/em\u003e (SALK_044831) and \u003cem\u003eatg1abct\u003c/em\u003e were obtained from Prof. Fa Qiang Li [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Other stable expression transgenic plants were generated in this study.\u003c/p\u003e \u003cp\u003eArabidopsis seedlings were surface sterilized and vernalized at 4℃ for 2 to 3 days, then germinated on 1/2 MS medium at 22℃ under an LD photoperiod (16 h light/8 h darkness) with illumination at ~\u0026thinsp;100 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2 s\u0026minus;1\u003c/sup\u003e. After 1 week, the seedlings were transferred to soil for further growth. \u003cem\u003eNicotiana benthamiana\u003c/em\u003e was subsequently grown under LD conditions. One-month-old tobacco plants were used for transient expression assays.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAssays for freezing tolerance, chlorophyll content, electrolyte leakage, and proline content\u003c/h3\u003e\n\u003cp\u003eFreezing tolerance was assessed as previously described [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Briefly, 10-day-old plants grown at 22\u0026deg;C on 1/2 MS plates were treated with or without cold acclimation (4\u0026deg;C for 3 d) and then subjected to a freezing assay. The program was set at 0\u0026deg;C, and 1\u0026deg;C h was dropped to the desired temperature. After freezing treatment, the plants were incubated at 4\u0026deg;C in the dark for 12 h and then transferred to 22\u0026deg;C for an additional 2\u0026ndash;3 d. Chlorophyll was extracted and measured as described [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Electrolyte leakage assays were performed as described [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. The proline content was measured as previously described [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. At least three independent experiments were performed, and each experiment was performed with three technical replicates.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChilling stress response assay and anthocyanin extraction\u003c/h2\u003e \u003cp\u003eFor chilling stress, 7-day-old wild-type, \u003cem\u003eatg1abct\u003c/em\u003e, \u003cem\u003eatg13ab\u003c/em\u003e, \u003cem\u003eatg7-3\u003c/em\u003e, and \u003cem\u003eatg5-1\u003c/em\u003e seedlings grown on 1/2 MS plates at 22\u0026deg;C were transferred to a 4\u0026deg;C growth chamber (16 h light/8 h darkness with illumination at ~\u0026thinsp;100 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2 s\u0026minus;1\u003c/sup\u003e) and maintained for the indicated times. At least three independent experiments were performed, and each experiment was performed with three technical replicates.\u003c/p\u003e \u003cp\u003eAnthocyanin measurement was performed as described previously [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Arabidopsis seedlings were incubated in extraction buffer (methanol containing 1% HCl) overnight at 4\u0026deg;C in the dark. The samples were centrifuged, and the supernatants were collected for absorbance quantification at 530 and 657 nm. (A530\u0026thinsp;\u0026minus;\u0026thinsp;0.25 \u0026times; A657) per gram fresh weight was used to quantify the relative amounts of anthocyanins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR analysis\u003c/h2\u003e \u003cp\u003eRNA extraction was performed according to the instructions of the total RNA kit (OMEGA). 5 \u0026times; PrimeScript\u0026trade; RT Master Mix (TAKARA) was used to synthesize cDNA. qRT-PCR analysis was performed using SYBR Premix Ex Taq II (TaKaRa) and a StepOne PCR instrument. The 2\u0026minus;\u003csup\u003e△△\u003c/sup\u003eCT method was used to calculate the relative expression of genes. \u003cem\u003eUbiquitin 10\u003c/em\u003e (\u003cem\u003eUBQ10\u003c/em\u003e) was used as a reference gene. The gene-specific primers used for qRT-PCR are listed in Supplemental Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGUS staining\u003c/h2\u003e \u003cp\u003eTransgenic plants harboring the proATG13a::GUS or proATG8e::GUS constructs were immediately immersed in GUS staining solution (10 mM EDTA disodium salt, 100 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.5 mM K\u003csub\u003e4\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e, 0.5 mM K\u003csub\u003e3\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e, 0.5 mg/ml X-gluc (B5285, Sigma), pH 7.0) after various durations of 4\u0026deg;C treatment, incubated for 5 h at 37\u0026deg;C, and then decolorized with 70% ethanol. The stained tissues were observed and photographed with a Zeiss Discover.v20 imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDrug treatment and confocal laser scanning microscopy\u003c/h2\u003e \u003cp\u003eFor concanavalin A (ConA) treatment, seedlings of \u003cem\u003eproATG8a::GFP-ATG8a\u003c/em\u003e/Col were first grown on 1/2 MS agar media for 5 days at 22\u0026deg;C. The sterile seedlings were then transferred to 1/2 MS liquid media and either continued culturing at 22\u0026deg;C for 12 h (control) or exposed to 4\u0026deg;C in a growth chamber for 12 h under a standard photoperiod (16 h light/8 h dark cycle with ~\u0026thinsp;100 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; light intensity). After treatment, the seedlings were allowed to recover for 6 h at the normal temperature (22\u0026deg;C). Subsequently, either DMSO (control) or 0.5 \u0026micro;M ConA was added to the liquid medium, followed by further incubation at 22\u0026deg;C for 4 h. Following incubation, the GFP labeled autophagosomes in the roots were visualized with a Nikon A1\u0026thinsp;+\u0026thinsp;confocal laser scanning microscopeusing 40 \u0026times; water objectives. Excitation was performed at 488 nm, emission was collected at 500\u0026ndash;530 nm. Three to four representative images in the root elongation zone were photographed per seedling, and the number of GFP-ATG8a puncta in each image was counted and averaged. A total of ten seedlings were observed per treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eProtein isolation and immunoblot analysis\u003c/h2\u003e \u003cp\u003eFor protein extraction, Arabidopsis samples were ground and homogenized in ice-cold extraction buffer (10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM EDTA pH 8.0; 10% Glycerol; 0.5% Triton X-100; 1 \u0026times; cocktail). Samples were incubated on ice for 10\u0026ndash;15 min and centrifuged at 4℃ for 10 min at 12,000 \u003cem\u003eg\u003c/em\u003e. The supernatant was used for electrophoresis. For immunoblot analysis, total proteins were subjected to SDS-PAGE and electrophoretically transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore). For ATG8a assay, total protein was separated by SDS-PAGE in the presence of 6 M urea. The antibodies used for protein blot analysis were against ATG8a (Abcam, ab77003, 1:1000), GFP (Abmart, M20004, 1:5000), and β-actin (CWBIO, CW0264, 1:5000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHigh-throughput mRNA sequencing analysis\u003c/h2\u003e \u003cp\u003eFourteen-day-old seedlings grown on 1/2 MS medium at 22\u0026deg;C were treated at 4\u0026deg;C for 0, 3 or 24 h. Total RNA was extracted. and 3 \u0026micro;g of RNA for each sample was used for library construction and subsequent RNA-deep sequencing on the Illumina HiSeq 2500 platform. RNA-Seq data were collected from two independent experiments. The adaptor sequences and low-quality sequence reads were removed from the data sets. Raw sequences were transformed into clean reads after data processing. These clean reads were then mapped to the reference genome sequence. Only reads with a perfect match or one mismatch were further analyzed and annotated on the basis of the reference genome. HISAT2 tools were used for mapping with the reference genome. Gene function was annotated on the basis of TAIR10. Differential expression analysis of two conditions/groups was performed via the DESeq2 R package (1.26.0). DESeq2 provides statistical routines for determining differential expression in digital gene expression data via a model based on the negative binomial distribution. The resulting \u003cem\u003eP\u003c/em\u003e values were adjusted via Benjamini and Hochberg\u0026rsquo;s approach for controlling the false discovery rate. Genes with FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 \u0026amp; |Log2(foldchange)| \u0026ge;1 found by DESeq2 were considered differentially expressed. Gene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) was implemented via the GOseq R package-based Wallenius non-central hyper-geometric distribution (Young et al, 2010), which can adjust for gene length bias in DEGs [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRecombinant protein expression and cell-free assay\u003c/h2\u003e \u003cp\u003eFor the expression of recombinant protein in prokaryotic cells, the CDSs of COR47 were cloned and inserted into the pMal-cRi (MBP tag) vector and transformed into the \u003cem\u003eE. coli\u003c/em\u003e strain Rosetta, MBP tagged proteins were purified using PurKine\u0026trade; MBP-Tag Dextrin Resin 6FF (BMR20206, Abbkine) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eFor the cell-free assay, 2000\u0026thinsp;\u0026minus;\u0026thinsp;400 mg of 7-day-old normally growing plants was collected, and total protein was extracted via protein degradation buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM DTT, and 5 mM ATP). Add 100 ng of purified MBP-COR47 protein, and the mixture was incubated at room temperature for 0, 15, 30, 45, or 60 minutes. Equal amounts of the reaction mixture were collected at each time point to detect MBP-COR47 expression using anti-MBP (Abmart, 15089-1-AP, 1:5000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTransient expression in Arabidopsis protoplasts and cold treatment\u003c/h2\u003e \u003cp\u003eTransient expression assays in Arabidopsis protoplasts were conducted essentially as described previously [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Briefly, equal amounts (100 \u0026micro;g) of the COR47-GFP plasmid were independently transformed into protoplasts derived from the wild-type and the \u003cem\u003eatg13ab\u003c/em\u003e mutant. After incubation at 22\u0026deg;C for 16 h to allow sufficient expression of the fusion protein, the protoplasts were subjected to 4\u0026deg;C treatment for varying durations. Total protein was then extracted and analyzed by immunoblotting with anti-GFP and anti-Actin antibodies.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we revealed that unlike its well-described positive roles in common stresses such as salt, drought, and submergence, autophagy functions as a negative regulator of cold stress responses in Arabidopsis. The autophagy-deficient mutants exhibited enhanced freezing tolerance regardless of cold acclimation. Under prolonged chilling conditions, autophagy positively regulates anthocyanin accumulation. Cold treatment significantly suppressed autophagic activity, as evidenced by the transcriptional downregulation of autophagy-related genes, reduced autophagosome formation, and decreased ATG8 protein abundance. Notably, cold-responsive genes were constitutively upregulated in the autophagy mutants, which is consistent with the persistent accumulation of the cold-regulated protein COR47. These findings collectively demonstrate the suppressive role of autophagy in low-temperature adaptation in Arabidopsis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANS anthocyanin synthase\u003c/p\u003e\n\u003cp\u003eATG AuTophaGy\u003c/p\u003e\n\u003cp\u003eB1L bypass1-like\u003c/p\u003e\n\u003cp\u003eCBF c-repeat binding factors\u003c/p\u003e\n\u003cp\u003eCHS chalcone synthase\u003c/p\u003e\n\u003cp\u003eConA concanavalin A\u003c/p\u003e\n\u003cp\u003eCOR47 cold-regulated 47\u003c/p\u003e\n\u003cp\u003eDAS days after sowing\u003c/p\u003e\n\u003cp\u003eDDF1 dwarf and delayed flowering 1\u003c/p\u003e\n\u003cp\u003eDEG differentially expressed gene\u003c/p\u003e\n\u003cp\u003eDEP differentially expressed protein\u003c/p\u003e\n\u003cp\u003eDFR dihydroflavonol 4-reductase\u003c/p\u003e\n\u003cp\u003eDREB dehydration-responsive element binding\u003c/p\u003e\n\u003cp\u003eGFP green fluorescent protein\u003c/p\u003e\n\u003cp\u003eGols3 galactinol synthase 3\u003c/p\u003e\n\u003cp\u003eGUS glucuronidas\u003c/p\u003e\n\u003cp\u003eLTI30 low-temperature-induced 30\u003c/p\u003e\n\u003cp\u003eNBR1 neighbor of BRCA1\u003c/p\u003e\n\u003cp\u003ePE phosphatidylethanolamine\u003c/p\u003e\n\u003cp\u003ePI3K Phosphatidylinositol 3-kinase\u003c/p\u003e\n\u003cp\u003ePI3P phosphatidylinositol 3-phosphate\u003c/p\u003e\n\u003cp\u003eRD29A responsive to desiccation 29a\u003c/p\u003e\n\u003cp\u003eSNARE soluble NSF attachment protein receptor\u003c/p\u003e\n\u003cp\u003eUPS ubiquitin-proteasome system\u003c/p\u003e\n\u003cp\u003eZAT12 zinc finger of \u003cem\u003eArabidopsis thaliana \u003c/em\u003e12\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data generated or analyzed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by the National Natural Science Foundation of China (32400246) and the Shaanxi Provincial Natural Science Fund (22JR5RA831).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study\u0026rsquo;s conception and design. Q.L.W designed the research, Q.L.W, Y.S.P, S.J.G, B.L carried out the experiments, Q.L.W wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. FaQiang Li (South China Agricultural University) for providing the\u0026nbsp;\u003cem\u003eatg1abct\u003c/em\u003e and \u003cem\u003eatg13ab\u0026nbsp;\u003c/em\u003emutants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenerative AI statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare that no generative AI was used in the creation of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDing Y, Shi Y, Yang S. Regulatory networks underlying plant responses and adaptation to cold stress. Annu Rev Genet. 2024;58(1):43-65.\u003c/li\u003e\n\u003cli\u003eKosov\u0026aacute; K, Ne\u0026scaron;porov\u0026aacute; T, V\u0026iacute;t\u0026aacute;mv\u0026aacute;s P, V\u0026iacute;t\u0026aacute;mv\u0026aacute;s J, Kl\u0026iacute;ma M, Ovesn\u0026aacute; J, Pr\u0026aacute;\u0026scaron;il IT. How to survive mild winters: Cold acclimation, deacclimation, and reacclimation in winter wheat and barley. Plant Physiol Biochem. 2025;220:109541.\u003c/li\u003e\n\u003cli\u003eRam\u0026oacute;n A, Esteves A, Villad\u0026oacute;niga C, Chalar C, Castro-Sowinski S. A general overview of the multifactorial adaptation to cold: Biochemical mechanisms and strategies. Braz J Microbiol. 2023;54:2259-2287.\u003c/li\u003e\n\u003cli\u003eFeng Y, Li Z, Kong X, Khan A, Ullah N, Zhang X. Plant coping with cold stress: Molecular and physiological adaptive mechanisms with future perspectives. Cells. 2025;14(2):110.\u003c/li\u003e\n\u003cli\u003eSun JL, Li JY, Wang MJ, Song ZT, Liu JX. Protein quality control in plant organelles: Current progress and future perspectives. Mol Plant. 2021;14:95-114.\u003c/li\u003e\n\u003cli\u003eSu T, Yang M, Wang P, Zhao Y, Ma C. Interplay between the ubiquitin proteasome system and ubiquitin-mediated autophagy in plants. Cells. 2020;9(10):2219.\u003c/li\u003e\n\u003cli\u003ePohl C, Dikic I. Cellular quality control by the ubiquitin-proteasome system and autophagy. Science. 2019;366:818-822.\u003c/li\u003e\n\u003cli\u003eZientara-Rytter K, Sirko A. To deliver or to degrade \u0026ndash; an interplay of the ubiquitin-proteasome system, autophagy and vesicular transport in plants. FEBS J. 2016; 283(19):3534-3555.\u003c/li\u003e\n\u003cli\u003eMichaeli S, Galili G, Genschik P, Fernie AR, Avin-Wittenberg T. Autophagy in plants \u0026ndash; what\u0026apos;s new on the menu? Trends Plant Sci. 2016;21:134-144.\u003c/li\u003e\n\u003cli\u003eMarshall RS, Vierstra RD. Autophagic degradation of the 26S proteasome is mediated by the dual ATG8/ubiquitin receptor RPN10 in Arabidopsis. Mol Cell. 2015;58:1053-1066.\u003c/li\u003e\n\u003cli\u003eGross AS, Raffeiner M, Zeng Y, \u0026Uuml;st\u0026uuml;n S, Dagdas Y. Autophagy in plant health and disease. Annu Rev Plant Biol. 2025. doi: 10.1146/annurev-arplant-060324-094912.\u003c/li\u003e\n\u003cli\u003eLiu Y, Bassham DC. Autophagy: Pathways for self-eating in plant cells. Annu Rev Plant Biol. 2012;63:215-237.\u003c/li\u003e\n\u003cli\u003eYoshimoto K, Ohsumi Y. Unveiling the molecular mechanisms of plant autophagy \u0026ndash; from autophagosomes to vacuoles in plants. Plant Cell Physiol. 2018;59:1337-1344.\u003c/li\u003e\n\u003cli\u003eLv X, Pu X, Qin G, Zhu T, Lin H. The roles of autophagy in development and stress responses in\u003cem\u003e Arabidopsis thaliana\u003c/em\u003e. Apoptosis. 2014;19:905-921.\u003c/li\u003e\n\u003cli\u003eYano K, Suzuki T, Moriyasu Y. Constitutive autophagy in plant root cells. Autophagy. 2007;3:360-362.\u003c/li\u003e\n\u003cli\u003eBassham DC. Function and regulation of macroautophagy in plants. Biochim Biophys Acta. 2009;1793:1397-1403.\u003c/li\u003e\n\u003cli\u003eYoshimoto K, Hanaoka H, Sato S, Kato T, Tabata S, Noda T, Ohsumi Y. Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy. Plant Cell. 2004;16:2967-2983.\u003c/li\u003e\n\u003cli\u003eChung T, Phillips AR, Vierstra RD. ATG8 lipidation and ATG8‐mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci. Plant J. 2010;62:483-493.\u003c/li\u003e\n\u003cli\u003eSuttangkakul A, Li F, Chung T, Vierstra RD. The ATG1/ATG13 protein kinase complex is both a regulator and a target of autophagic recycling in Arabidopsis. Plant Cell. 2011;23:3761-3779.\u003c/li\u003e\n\u003cli\u003eNaumann C, M\u0026uuml;ller J, Sakhonwasee S, Wieghaus A, Hause G, Heisters M, B\u0026uuml;rstenbinder K, Abel S. The local phosphate deficiency response activates endoplasmic reticulum stress dependent autophagy. Plant Physiol. 2019;179:460-476.\u003c/li\u003e\n\u003cli\u003eLornac A, Hav\u0026eacute; M, Chardon F, Soulay F, Cl\u0026eacute;ment G, Avice JC, Masclaux-Daubresse C. Autophagy controls sulphur metabolism in the rosette leaves of Arabidopsis and facilitates S remobilization to the seeds. Cells. 2020;9(2):332.\u003c/li\u003e\n\u003cli\u003eLiu Y, Xiong Y, Bassham DC. Autophagy is required for tolerance of drought and salt stress in plants. Autophagy. 2009;5:954-963.\u003c/li\u003e\n\u003cli\u003eLuo L, Zhang P, Zhu R, Fu J, Su J, Zheng J, Wang Z, Wang D, Gong Q. Autophagy is rapidly induced by salt stress and is required for salt tolerance in Arabidopsis. Front Plant Sci. 2017;8:1459.\u003c/li\u003e\n\u003cli\u003eBao Y, Song W-M, Wang P, Yu X, Li B, Jiang C, Shiu S-H, Zhang H, Bassham DC. COST1 regulates autophagy to control plant drought tolerance. Pro Natl Acad Sci. 2020;117(13):7482-7493.\u003c/li\u003e\n\u003cli\u003eChen L, Liao B, Qi H, Xie LJ, Huang L, Tan WJ, Zhai N, Yuan LB, Zhou Y, Yu LJ, Chen QF, Shu W, Xiao S. Autophagy contributes to regulation of the hypoxia response during submergence in\u003cem\u003e Arabidopsis thaliana\u003c/em\u003e. Autophagy. 2015;11:2233-2246.\u003c/li\u003e\n\u003cli\u003eThirumalaikumar VP, Gorka M, Schulz K, Masclaux-Daubresse C, Sampathkumar A, Skirycz A, Vierstra RD, Balazadeh S. Selective autophagy regulates heat stress memory in Arabidopsis by NBR1-mediated targeting of HSP90.1 and ROF1. Autophagy. 2021;17:2184-2199.\u003c/li\u003e\n\u003cli\u003eSedaghatmehr M, Thirumalaikumar VP, Kamranfar I, Marmagne A, Masclaux-Daubresse C, Balazadeh S. A regulatory role of autophagy for resetting the memory of heat stress in plants. Plant Cell Environ. 2019;42:1054-1064.\u003c/li\u003e\n\u003cli\u003eZhai Y, Guo M, Wang H, Lu J, Liu J, Zhang C, Gong Z, Lu M. Autophagy, a conserved mechanism for protein degradation, responds to heat, and other abiotic stresses in \u003cem\u003eCapsicum annuum L.\u003c/em\u003e Front Plant Sci. 2016;7:131.\u003c/li\u003e\n\u003cli\u003eChi C, Li X, Fang P, Xia X, Shi K, Zhou Y, Zhou J, Yu J. Brassinosteroids act as a positive regulator of NBR1-dependent selective autophagy in response to chilling stress in tomato. J Exp Bot. 2020;71:1092-1106.\u003c/li\u003e\n\u003cli\u003eLi Q, Wang B, Yu H. New mechanism of strigolactone-regulated cold tolerance in tomato. New Phytol. 2025;245:921-923.\u003c/li\u003e\n\u003cli\u003eChen XL, Zheng XL, Xu T, Zou JP, Jin WD, Wang GH, Yang P, Zhou J. Role of selective autophagy receptors in tomato response to cold stress. Environ Exp Bot. 2023;213:105426.\u003c/li\u003e\n\u003cli\u003eZhao W, Song J, Wang M, Chen X, Du B, An Y, Zhang L, Wang D, Guo C. Alfalfa MsATG13 confers cold stress tolerance to plants by promoting autophagy. Int J Mol Sci. 2023;24(15):12033.\u003c/li\u003e\n\u003cli\u003eUsadel B, Bl\u0026auml;sing OE, Gibon Y, Poree F, H\u0026ouml;hne M, G\u0026uuml;nter M, Trethewey R, Kamlage B, Poorter H, Stitt M. Multilevel genomic analysis of the response of transcripts, enzyme activities and metabolites in Arabidopsis rosettes to a progressive decrease of temperature in the non-freezing range. Plant Cell Environ. 2008;31:518-547.\u003c/li\u003e\n\u003cli\u003eKlińska-Bąchor S, Kędzierska S, Demski K, Banaś A. Phospholipid: Diacylglycerol acyltransferase1-overexpression stimulates lipid turnover, oil production and fitness in cold-grown plants. BMC Plant Biol. 2023;23:370.\u003c/li\u003e\n\u003cli\u003eSato A, Inayoshi S, Kitawaki K, Mihara R, Yoneda K, Ito-Inaba Y, Inaba T. Autophagy is suppressed by low temperatures and is dispensable for cold acclimation in Arabidopsis. Physiol Plantarum. 2024;176:e14409.\u003c/li\u003e\n\u003cli\u003eHuang X, Zheng C, Liu F, Yang C, Zheng P, Lu X, Tian J, Chung T, Otegui MS, Xiao S, Gao C, Vierstra RD, Li F. Genetic analyses of the Arabidopsis ATG1 kinase complex reveal both kinase-dependent and independent autophagic routes during fixed-carbon starvation. Plant Cell. 2019;31:2973-2995.\u003c/li\u003e\n\u003cli\u003eGhosh UK, Islam MN, Siddiqui MN, Cao X, Khan MAR. Proline, a multifaceted signalling molecule in plant responses to abiotic stress: Understanding the physiological mechanisms. Plant Biol. 2022;24(2):227-239.\u003c/li\u003e\n\u003cli\u003eSchulz E, Tohge T, Zuther E, Fernie AR, Hincha DK. Natural variation in flavonol and anthocyanin metabolism during cold acclimation in \u003cem\u003eArabidopsis thaliana \u003c/em\u003eaccessions. Plant Cell Environ. 2015;38(8):1658-1672.\u003c/li\u003e\n\u003cli\u003eZhou H, He J, Zhang Y, Zhao H, Sun X, Chen X, Liu X, Zheng Y, Lin H. RHA2b-mediated MYB30 degradation facilitates MYB75-regulated, sucrose-induced anthocyanin biosynthesis in Arabidopsis seedlings. Plant Commun. 2024;5:100744.\u003c/li\u003e\n\u003cli\u003eXie Y, Tan H, Ma Z, Huang J. DELLA proteins promote anthocyanin biosynthesis via sequestering MYBL2 and JAZ suppressors of the MYB/bHLH/WD40 complex in\u003cem\u003e Arabidopsis thaliana\u003c/em\u003e. Mol Plant. 2016;9:711-721.\u003c/li\u003e\n\u003cli\u003eMasclaux-Daubresse C, Cl\u0026eacute;ment G, Anne P, Routaboul J-M, Guiboileau A, Soulay F, Shirasu K, Yoshimoto K. Stitching together the multiple dimensions of autophagy using metabolomics and transcriptomics reveals impacts on metabolism, development, and plant responses to the environment in Arabidopsis. Plant Cell. 2014;26(5):1857-1877.\u003c/li\u003e\n\u003cli\u003eXiao S, Gao W, Chen QF, Chan SW, Zheng SX, Ma J, Wang M, Welti R, Chye ML. Overexpression of Arabidopsis acyl-CoA binding protein ACBP3 promotes starvation-induced and age-dependent leaf senescence. Plant Cell. 2010;22:1463-1482.\u003c/li\u003e\n\u003cli\u003eQi H, Wang Y, Bao Y, Bassham DC, Chen L, Chen QF, Hou S, Hwang I, Huang L, Lai Z, Li F, Liu Y, Qiu R, Wang H, Wang P, Xie Q, Zeng Y, Zhuang X, Gao C, Jiang L, Xiao S. Studying plant autophagy: Challenges and recommended methodologies. Adv Biotechnol. 2023;1:2.\u003c/li\u003e\n\u003cli\u003eKang HG, Kim J, Kim B, Jeong H, Choi SH, Kim EK, Lee HY, Lim PO. Overexpression of FTL1/DDF1, an AP2 transcription factor, enhances tolerance to cold, drought, and heat stresses in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Plant Sci. 2011;180:634-641.\u003c/li\u003e\n\u003cli\u003eJia Y, Ding Y, Shi Y, Zhang X, Gong Z, Yang S. The cbfs triple mutants reveal the essential functions of CBFs in cold acclimation and allow the definition of CBF regulons in Arabidopsis. New Phytol. 2016;212:345-353.\u003c/li\u003e\n\u003cli\u003eChen T, Chen JH, Zhang W, Yang G, Yu LJ, Li DM, Li B, Sheng HM, Zhang H, An LZ. BYPASS1-LIKE, a DUF793 family protein, participates in freezing tolerance via the CBF pathway in Arabidopsis. Front Plant Sci. 2019;10:807.\u003c/li\u003e\n\u003cli\u003eGismondi M, Strologo L, Gabilondo J, Budde C, Drincovich MF, Bustamante C. Characterization of ZAT12 protein from \u003cem\u003ePrunus persica\u003c/em\u003e: role in fruit chilling injury tolerance and identification of gene targets. Planta. 2024;261:14.\u003c/li\u003e\n\u003cli\u003eZhang L, Jiang X, Liu Q, Ahammed GJ, Lin R, Wang L, Shao S, Yu J, Zhou Y. The HY5 and MYB15 transcription factors positively regulate cold tolerance in tomato via the CBF pathway. Plant Cell Environ. 2020;43:2712-2726.\u003c/li\u003e\n\u003cli\u003eCho Y, Kwon H, Kim BC, Shim D, Ha J. Identification of genetic factors influencing flavonoid biosynthesis through pooled transcriptome analysis in mungbean sprouts. Front Plant Sci. 2025;16:1540674.\u003c/li\u003e\n\u003cli\u003eLi S, Wang W, Gao J, Yin K, Wang R, Wang C, Petersen M, Mundy J, Qiu JL. MYB75 phosphorylation by MPK4 is required for light-induced anthocyanin accumulation in Arabidopsis. Plant Cell. 2016, 28(11):2866-2883.\u003c/li\u003e\n\u003cli\u003eLi S, He L, Yang Y, Zhang Y, Han X, Hu Y, Jiang Y. INDUCER OF CBF EXPRESSION 1 promotes cold-enhanced immunity by directly activating salicylic acid signaling. Plant Cell. 2024;36(7):2587-2606.\u003c/li\u003e\n\u003cli\u003eKidokoro S, Shinozaki K, Yamaguchi-Shinozaki K. Transcriptional regulatory network of plant cold-stress responses. Trends Plant Sci. 2022;27:922-935.\u003c/li\u003e\n\u003cli\u003eCheng S, Fan S, Yang C, Hu W, Liu F. Proteomics revealed novel functions and drought tolerance of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e protein kinase ATG1. BMC Plant Biol. 2025;23:48.\u003c/li\u003e\n\u003cli\u003eLi L, Lee CP, Ding X, Qin Y, Wijerathna-Yapa A, Broda M, Otegui MS, Millar AH. Defects in autophagy lead to selective in vivo changes in turnover of cytosolic and organelle proteins in Arabidopsis. Plant Cell. 2022;34:3936-3960.\u003c/li\u003e\n\u003cli\u003eHern\u0026aacute;ndez-S\u0026aacute;nchez IE, Maruri-L\u0026oacute;pez I, Graether SP, Jim\u0026eacute;nez-Bremont JF. In vivo evidence for homo- and heterodimeric interactions of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e dehydrins AtCOR47, AtERD10, and AtRAB18. Sci Rep. 2017;7(1):17036.\u003c/li\u003e\n\u003cli\u003eAvin-Wittenberg T. Autophagy and its role in plant abiotic stress management. Plant Cell Environ. 2019;42(3):1045-1053.\u003c/li\u003e\n\u003cli\u003eYagyu M, Yoshimoto K. New insights into plant autophagy: molecular mechanisms and roles in development and stress responses. J Exp Bot. 2024;75:1234-1251.\u003c/li\u003e\n\u003cli\u003ePourcel L, Irani NG, Lu Y, Riedl K, Schwartz S, Grotewold E. The formation of anthocyanic vacuolar inclusions in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and implications for the sequestration of anthocyanin pigments. Mol Plant. 2010;3(1):78-90.\u003c/li\u003e\n\u003cli\u003eYang C, Luo M, Zhuang X, Li F, Gao C. Transcriptional and epigenetic regulation of autophagy in plants. Trends Genet. 2020;36(9):676-688\u003c/li\u003e\n\u003cli\u003eQuinn PJ. Effects of temperature on cell membranes. Symp Soc Exp Biol. 1988;42:237-258.\u003c/li\u003e\n\u003cli\u003eLiu H, Wang FF, Peng XJ, Huang JH, Shen SH. Global phosphoproteomic analysis reveals the defense and response mechanisms of jatropha curcas seedling under chilling stress. Int J Mol Sci. 2019;20(1):208.\u003c/li\u003e\n\u003cli\u003eLi X, Cai J, Liu F, Dai T, Cao W, Jiang D. Cold priming drives the sub-cellular antioxidant systems to protect photosynthetic electron transport against subsequent low temperature stress in winter wheat. Plant Physiol Biochem. 2014;82:34-43.\u003c/li\u003e\n\u003cli\u003eWu Z, Han S, Zhou H, Tuang ZK, Wang Y, Jin Y, Shi H, Yang W. Cold stress activates disease resistance in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e through a salicylic acid dependent pathway. Plant Cell Environ. 2019;42(9):2645-2663.\u003c/li\u003e\n\u003cli\u003evan Hulten M, Pelser M, van Loon LC, Pieterse CM, Ton J: Costs and benefits of priming for defense in Arabidopsis. Proc Natl Acad Sci. 2006;103(14):5602-5607.\u003c/li\u003e\n\u003cli\u003eGuo W, Ward RW, Thomashow MF. Characterization of a cold-regulated wheat gene related to Arabidopsis COR47. Plant Physiol. 1992;100:915-922.\u003c/li\u003e\n\u003cli\u003eSzlachtowska Z, Rurek M. Plant dehydrins and dehydrin-like proteins: Characterization and participation in abiotic stress response. Front Plant Sci. 2023;14:1213188.\u003c/li\u003e\n\u003cli\u003eRorat T. Plant dehydrins-tissue location, structure and function. Cell Mol Biol Lett. 2006;11:536-556.\u003c/li\u003e\n\u003cli\u003eLi X, Liu Q, Feng H, Deng J, Zhang R, Wen J, Dong J, Wang T. Dehydrin MtCAS31 promotes autophagic degradation under drought stress. Autophagy. 2020;16:862-877.\u003c/li\u003e\n\u003cli\u003eWang X, Zhang X, Song CP, Gong Z, Yang S, Ding Y. PUB25 and PUB26 dynamically modulate ICE1 stability via differential ubiquitination during cold stress in Arabidopsis. Plant Cell. 2023;35:3585-3603.\u003c/li\u003e\n\u003cli\u003eWang Q, Qin Q, Su M, Li N, Zhang J, Liu Y, Yan L, Hou S. Type one protein phosphatase regulates fixed-carbon starvation-induced autophagy in Arabidopsis. Plant Cell. 2022;34(11):4531-4553.\u003c/li\u003e\n\u003cli\u003eYoung MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: Accounting for selection bias. Genome Biol. 2010;11:R14.\u003c/li\u003e\n\u003cli\u003eMiao Y, Jiang L. Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells. Nat Protoc. 2007;2, 2348-2353.\u003c/li\u003e\n\u003cli\u003eZhuang X, Wang H, Lam SK, Gao C, Wang X, Cai Y, Jiang L. A BAR-domain protein SH3P2, which binds to phosphatidylinositol 3-phosphate and ATG8, regulates autophagosome formation in Arabidopsis. Plant Cell. 2013;25:4596-4615.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Autophagy, Arabidopsis, cold stress, atg13ab, ATG8, COR47","lastPublishedDoi":"10.21203/rs.3.rs-6620529/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6620529/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePlants have evolved multiple strategies to cope with the ever-changing external environment. Autophagy, as one of the crucial mechanisms involved, has been demonstrated to play a pivotal role in plant responses and adaptation to abiotic stresses. However, the precise molecular mechanisms underlying the role of autophagy in mediating cold stress remain to be fully elucidated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we demonstrated that autophagy mutants presented increased freezing tolerance under both non-acclimated and cold-acclimated conditions. Autophagy positively regulates the expression of anthocyanin biosynthesis-related genes, thereby influencing anthocyanin accumulation in Arabidopsis under low-temperature conditions. Moreover, we found that cold stress directly suppresses the expression of autophagy-related genes and reduces autophagic flux. The RNA-seq data revealed that cold-responsive genes were pre-activated in the autophagy mutant \u003cem\u003eatg13ab\u003c/em\u003e even before cold treatment. Additionally, we observed constitutive accumulation of the dehydrin protein COR47 in \u003cem\u003eatg13ab\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTaken together, these data suggest that autophagy is a negative regulator of freezing tolerance in Arabidopsis.\u003c/p\u003e","manuscriptTitle":"Inhibition of Autophagy Increases Freezing Survival in Arabidopsis thaliana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-09 08:12:06","doi":"10.21203/rs.3.rs-6620529/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-14T06:50:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-13T13:20:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-13T13:16:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-05-08T11:57:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b61e9200-bac9-43b6-b723-cdd3758c3048","owner":[],"postedDate":"May 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T16:39:46+00:00","versionOfRecord":{"articleIdentity":"rs-6620529","link":"https://doi.org/10.1186/s12870-025-07066-9","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-07-30 16:20:56","publishedOnDateReadable":"July 30th, 2025"},"versionCreatedAt":"2025-05-09 08:12:06","video":"","vorDoi":"10.1186/s12870-025-07066-9","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07066-9","workflowStages":[]},"version":"v1","identity":"rs-6620529","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6620529","identity":"rs-6620529","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}