Autophagy Is More Vital in Tomato and Citrus than in Arabidopsis

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While its core machinery is conserved across species, the differential regulation of autophagy in annual versus perennial plants, particularly with respect to resource allocation trade-offs, remains poorly understood. Here, we conducted a comparative study to investigate the function of autophagy in horticultural plants with different life cycles. Using autophagy-deficient materials targeting ATG7 in tomato (Solanum lycopersicum) and citrus (Fortunella hindsii), alongside Arabidopsis as a reference, we analysed the impact of autophagy on vegetative growth, reproductive development, and nutrient stress responses in annual and perennial plants. Autophagy deficiency consistently impaired growth across species, but F. hindsii exhibited more severe growth inhibition and premature leaf senescence, highlighting life-history-dependent differences in autophagy reliance. Comparative transcriptomic analysis of F. hindsii further revealed potential molecular networks underlying autophagy deficiency-induced leaf senescence. In conclusion, our study provides the first evidence on how autophagy differentially underpins annual and perennial life strategies, offering a theoretical foundation for future autophagy-based breeding approaches. Autophagy Tomato Citrus Plant development Leaf senescence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Plants exhibit remarkable diversity in strategies to adapt to vastly different environments on Earth. Their life cycles exhibit two distinct patterns: annual plants tend to reproduce once and then die, while perennial plants engage in long-term trade-offs among growth, reproduction, and survival (Lundgren and Des Marais 2020). Therefore, material cycling and energy utilisation within plants are likely different. Autophagy is a highly conserved process in eukaryotes that degrades and recycles cytoplasmic components, including proteins, protein aggregates, and organelles, during development or under environmental stress. In plants, two main types of autophagy have been described: microautophagy and macroautophagy (Ding et al 2018; Petersen et al 2024). In microautophagy, cytoplasmic components are directly engulfed by the vacuole through invagination of the tonoplast. While in macroautophagy, a cup-shaped membrane structure called the phagophore forms around the cargo, expands, and eventually seals into a double-membrane vesicle known as an autophagosome. Subsequently, the outer membrane of the autophagosome fuses with the tonoplast, releasing the autophagic body into the vacuole for degradation (Avin-Wittenberg et al 2018; Marshall and Vierstra 2018). Autophagy is initiated by the ATG1 kinase complex. This complex, comprising the catalytic ATG1 subunit along with regulatory subunits ATG13, ATG11, and ATG101, phosphorylates and activates downstream autophagy components upon its activation (Kamada et al 2000). The phosphatidylinositol 3-kinase (PI3K) complex generates phosphatidylinositol 3-phosphate (PI3P) at the phagophore (Kihara et al 2001), which in turn recruits PI3P-binding proteins such as ATG18 (Proikas-Cezanne et al 2007). Phagophore expansion and maturation depend on lipid delivery mediated by the lipid scramblase ATG9 and the lipid transfer protein ATG2 (Matoba et al 2020; Valverde et al 2019), as well as two ubiquitin-like conjugation cascades that ultimately lead to the attachment of the ubiquitin-like protein ATG8 to phosphatidylethanolamine (PE) on the autophagosome membrane (Ichimura et al 2000). ATG5 and ATG7 are widely recognised as essential regulators of ATG8 lipidation, a landmark process in autophagosome formation and autophagic flux (Ichimura et al 2000; Kim et al 1999; Mizushima et al 1998). In most organisms, interfering with the function of key ATG genes is sufficient to block autophagy activity and induce autophagy-defective phenotypes (Kurusu et al 2014; Tang and Bassham 2018). However, while the core regulatory mechanisms of autophagy have been well-characterised in model plants (e.g., Arabidopsis thaliana ) and major crops (e.g., rice, Oryza sativa ), research on autophagy in horticultural crops remains in its infancy. In this study, we investigated two contrasting horticultural systems: tomato ( Solanum lycopersicum ) and mini citrus ( Fortunella hindsii ), as examples for annual and perennial crops, respectively (Zhu et al 2019). We performed comparative phenotype studies using the Arabidopsis atg7 T-DNA knock-out mutant, and the previously generated ATG7 -RNAi lines of tomato and citrus (Guo et al 2025a; Guo et al 2025b), and found that autophagy deficiency consistently impairs vegetative growth across all three species. Notably, in the perennial citrus plants ( F. hindsii ), the reduction in biomass exhibited a cumulative effect, resulting in progressively more stunted growth over multiple years. Regarding reproductive development, all three autophagy-deficient genotypes exhibited reduced seed production. Under dark-induced carbon starvation, both Arabidopsis atg7 mutants and SlATG7- RNAi lines exhibited premature leaf senescence. Strikingly, the FhATG7 -RNAi lines displayed a pronounced premature leaf senescence phenotype even under normal growth conditions. To investigate the underlying cause of premature leaf senescence in these autophagy-deficient materials, we measured the reactive oxygen species (ROS) content and found that FhATG7 -RNAi leaves exhibited increased ROS levels. Comparative transcriptome analysis further revealed that knockdown of the FhATG7 gene disrupts autophagic flux, leading to the activation of senescence-related hormonal signalling pathways such as ABA, ethylene, and jasmonic acid, thereby triggering systemic senescence initiation at the cellular level. Collectively, this work elucidates autophagy-dependent developmental regulation in horticultural crops, with particular significance for perennial species, and offers a valuable foundation for targeted breeding strategies to enhance sustainable productivity and stress adaptation in perennial horticultural crops. Materials and methods Plant materials and growth conditions All Arabidopsis lines used in this study are of the Columbia-0 (Col-0) background. The atg7 (SAIL_11_H07) mutants have been described previously(Guan et al 2019). The Arabidopsis seeds were surface sterilised and germinated on half Murashige and Skoog (MS) medium containing 0.8% (w/v) agar and 3% (w/v) sucrose, pH 5.8. Seedlings were cultured in a plant growth chamber at 22 °C under a 16-h light and 8-h dark cycle before being transferred into soil in a plant growth room. The FhATG7 -RNAi transgenic citrus ( Fortunella hindsii ) has been described previously (Guo et al 2025b). The citrus plants were grown in the greenhouse of the Citrus Cultivation Centre at Huazhong Agricultural University (Wuhan, China) under standard cultivation conditions. The SlATG7 -RNAi transgenic tomato ( Solanum lycopersicum, Micro-Tom) has been described previously (Guo et al 2025a). The tomato plants were grown under standard greenhouse conditions (14 h of light at 26°C; 10 h dark at 20°C). Identification of ATG genes in tomato and mini citrus. Homology searches were performed against the tomato(https://solgenomics.net) and mini Citrus (http://citrus.hzau.edu.cn) genomes using conserved domains from yeast ATG proteins. Simultaneously, amino acid sequences of Arabidopsis ATG proteins were retrieved from the TAIR database (https://www.arabidopsis.org/) and used as queries for BLASTp searches (E‑value ≤ 1 × 10⁻⁵) in the tomato and mini citrus genomes. Candidate ATG protein sequences were further validated using the NCBI Conserved Domain Database (CDD; https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to confirm the presence of ATG protein domains. Carbon deprivation treatment. Arabidopsis seeds were sown on 1/2 MS plates and grown under a 16-h light/8-h dark photoperiod at 22°C. For carbon deprivation treatment, 7-day-old seedlings were transferred onto 1/2 MS plates without sucrose, wrapped in aluminium foil to maintain darkness, and then returned to the same growth chamber for the indicated durations. Seedlings harvested immediately after transfer served as the 0‑day carbon deprivation control (Xue et al 2022). For carbon deprivation of tomato leaves, leaves were excised with scissors, placed in distilled water, and then incubated in darkness for 7 days before sampling. Chlorophyll content measurement. Fresh leaf samples (0.2g) were weighed into a 10-mL tube and cut with scissors, then 10 mL of extraction buffer (acetone-ethanol, 1:1, v/v) was added. After 24 h of darkness treatment, the absorbance at 645 and 663 nm was measured using a microplate reader (BioTek Synergy H1), and chlorophyll content was determined using the equation of Lichtenthaler (1987). ROS Visualization Mature leaves from WT and FhATG7 -RNAi lines of Fortunella hindsii were collected. The leaves were incubated in the dark for 30 minutes with 50 µM of the reactive oxygen species (ROS)-specific fluorescent probe H2DCFDA. Subsequently, the leaves were removed from the incubation solution, thoroughly rinsed, and observed using an in vivo imaging system (NightSHADE L985, Berthold) with excitation/emission wavelengths set at 480 nm/520 nm. Total RNA extraction and transcriptome sequencing A total of four sample groups were selected for this study, encompassing young leaves and mature leaves from both wild-type and FhATG7 -RNAi transgenic lines. Each group consisted of three biological replicates. Total RNA was extracted from leaf tissues using a column-based plant RNA extraction kit. The quality and integrity of RNA samples were assessed using 1.0% agarose gel electrophoresis, a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). For transcriptome sequencing, RNA-seq libraries were prepared using the NEB Next Ultra II RNA Library Prep Kit for Illumina (New England Biolabs Inc., Ipswich, Massachusetts, USA) following the manufacturer's (Illumina) instructions and were subsequently sequenced on the Illumina Novaseq 6000 platform (Personal Biotechnology Co., Ltd, China). Differential expression gene analysis and GO-term pathway enrichment analysis The Raw RNA-seq reads were first subjected to quality control using Fastp to remove adapter sequences and low-quality bases. The clean reads were then aligned to the F. hindsii reference genome using HISAT2. Gene expression levels were quantified via feature Counts and normalised as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Differential expression analysis was performed using the DESeq2 package in R, with genes showing an adjusted *p*-value (FDR) < 0.05 and an absolute log2 fold change ≥ 1 considered significantly differentially expressed. Gene Ontology (GO) enrichment analysis for these significant genes was conducted using the clusterProfiler R package, with terms considered enriched at FDR < 0.05. Statistical analysis All statistical graphs were performed using GraphPad Prism 9 software. The results were compared using appropriate statistical analysis methods (Tukey’s test, one-way ANOVA or two-way ANOVA for multiple comparisons), and data were expressed as the mean ± SEM. A p-value below 0.05 was considered significant. Results Identification and Characterisation of ATG genes in tomato and mini citrus ATG genes play crucial roles in autophagy. To investigate the functions of autophagy-related genes in annual and perennial plants, we identified the number of key ATG genes in the genomes of tomato ( Solanum lycopersicum ) and citrus ( Fortunella hindsii ) (Table S1). Similar to Arabidopsis, ATG1 , ATG8 , and ATG18 are consistently encoded by small gene families across all three species, whereas the number of other ATG genes varies between species. In Arabidopsis and other annual crops, mutants of ATG genes commonly exhibit characteristic phenotypes such as reduced growth, premature senescence, decreased seed yield, and enhanced sensitivity to abiotic stress (Petersen et al 2024). We selected the ATG7 gene as the example and conducted a systematic comparison of autophagy-deficient materials from the perennial plant F. hindsii and from the annual model plant Arabidopsis, as well as from tomato. The Role of Autophagy in Vegetative Growth The expression pattern indicates that ATG7 is essential in both tomato and citrus (Fig. S1), required to maintain basal autophagy and to play essential roles throughout all developmental stages. To generate autophagy-deficient mutants, we knocked out SlATG7 in tomato via CRISPR-Cas9 gene editing. Interestingly, in contrast to the Arabidopsis atg7 loss-of-function mutant, which develops and sets seeds normally, homozygous atg7 knockout in tomato resulted in a lethal phenotype (Fig. S2). These results suggest that autophagy plays differential levels of importance across species. Compared with Arabidopsis, tomato has greater biomass and produces fleshy fruits, which likely impose higher energy demands. We therefore adopted RNA interference (RNAi) and successfully generated transgenic RNAi lines targeting SlATG7 in tomato and FhATG7 in citrus, confirming a significant reduction in autophagy activity in these lines (Guo et al 2025a; Guo et al 2025b). In Arabidopsis thaliana , 7-day-old atg7 mutants exhibited slightly shorter roots than the wild type under normal growth conditions (Fig. 1ab). Following transplantation into soil, the inflorescence height was also reduced (Fig. 1cd). These results indicate that autophagy deficiency reduces vegetative growth in Arabidopsis. Correspondingly, SlATG7 -RNAi transgenic tomato lines also displayed pronounced developmental defects during vegetative growth. Hypocotyl length in seedlings of two independent transgenic lines was significantly shorter than that of the wild type, indicating growth inhibition (Fig.1e). Further observation at the flowering stage revealed that the plant height of SlATG7 -RNAi lines was significantly lower than that of the WT (Fig.1fh). These results demonstrate that reduced autophagy levels in tomato significantly inhibit growth. In citrus, continuous three-year observation of FhATG7 ‑RNAi T 1 seedlings revealed that the growth of two transgenic lines was consistently and significantly lower than that of the WT (Fig. 2a–c). Statistical analysis of plant height indicated that the difference became more pronounced in two‑ and three‑year‑old plants (Fig. 2d). In this perennial species, autophagy deficiency likely leads to reduced plant growth, and such impairment may accumulate over successive growing seasons, resulting in progressively more marked phenotypic divergence. In summary, autophagy deficiency consistently suppresses vegetative growth in Arabidopsis, tomato, and mini citrus, underscoring the conserved role of autophagy in maintaining basic plant growth capacity. However, the phenotypic severity and temporal pattern of this inhibition exhibit species-specific variation. Complete knockout of ATG7 leads to only mild growth defects in Arabidopsis but causes lethality in tomato. In contrast, knockdown of ATG7 expression level is sufficient to induce significant growth reduction in both tomato and mini citrus. Furthermore, in perennial citrus plants, autophagy deficiency results in cumulative growth impairment, with phenotypic divergence from the wild type progressively widening over successive years. These findings indicate that the functional importance of autophagy varies across species and that, in perennial plants, autophagy not only regulates growth within a single season but may also play a critical role in interannual resource allocation and long-term survival. The Role of Autophagy in Reproductive Growth In Arabidopsis atg7 mutants, siliques exhibited significantly higher seed abortion rates than in the WT (Fig. 3a-c). In tomato SlATG7 -RNAi lines, no significant differences were observed in overall fruit morphology or individual fruit weight (Fig. S3). However, a marked reduction in the number of seeds per fruit was evident. Furthermore, the seeds exhibited a rough surface and developed brown patches on the seed coat in the dry state, in contrast to the smooth surface and whitish coat colour of WT seeds. Seed size measurements indicated a significant increase in seed length in SlATG7 -RNAi lines (Fig. 3d-g). These results confirm that SlATG7 -RNAi leads to substantial alterations in both seed quantity and morphology. In citrus, FhATG7 -RNAi lines exhibited significantly compromised vegetative growth, leading to a marked reduction in flower production (Fig. 3k). The number of fruits per plant was quantified, revealing a significant decrease in the FhATG7 -RNAi lines compared to the WT control (Fig. 3hi). Seeds of the transgenic lines also displayed obvious developmental abnormalities (Fig. 3j). These findings suggest that the effect of autophagy on reproductive development is closely tied to the differences between annual and perennial plants. In annual species that invest resources into a single major reproductive event, autophagy deficiency directly impairs the final stage of seed production. In contrast, perennial plants must balance multi‑year cycles of vegetative growth and reproductive allocation. Here, autophagy deficiency primarily reduces overall plant vigour, limiting reproductive potential at the source through decreased flower number, resulting in cumulative declines in reproductive success across seasons. Autophagy in Response to Nutrient Stress in Horticultural Crops In higher plants, modulation of nutrient availability—such as deprivation of carbon or nitrogen sources—serves as a key regulator of senescence progression (Li and Vierstra 2012; Liu and Bassham 2012). We examined leaf senescence under both nutrient-sufficient and carbon-deficient conditions by measuring chlorophyll content as an indicator of senescence. In Arabidopsis atg7 mutants, no significant difference compared with the wild type was observed under nutrient-sufficient conditions (Fig.4a). However, under carbon-deficient stress, the atg7 mutant exhibited pronounced senescence (Fig.4ab). In tomato, SlATG7 -RNAi lines did not show significant early senescence during the vegetative growth stage (Fig.4c). When tomato leaves were subjected to carbon-deficient stress, leaves of the SlATG7 -RNAi lines turned yellow and chlorophyll content decreased significantly (Fig.4cd). In the perennial mini citrus, the FhATG7 -RNAi lines exhibited obvious premature leaf senescence even under normal growth conditions. We measured chlorophyll content in leaves collected from branches at comparable positions, and the results showed a significant decrease compared with the wild type (Fig. 4ef). These suggest that the role of autophagy in plant responses to nutrient stress may differ between annual and perennial plants. Annual plants likely rely more heavily on autophagy for rapid resource reallocation during short-term, acute stress to sustain survival. In contrast, perennial plants, due to their long-lived nature, require autophagy to continuously maintain leaf protein homeostasis and nutrient balance under normal conditions, thereby supporting the functional longevity of their perennial organs (e.g., evergreen leaves). Consequently, the disruption of autophagy imposes a more severe homeostatic imbalance in perennial plants, which is sufficient to trigger senescence programs even in the absence of external stress. RNA-Seq Analysis of Leaf Transcriptome in FhATG7 -RNAi Lines To investigate the causes of premature leaf senescence in the FhATG7 -RNAi line, transcriptome sequencing was performed on young and mature leaves from both the FhATG7 -RNAi line and WT (Fig. 5a). The analysis revealed that 7,804 differentially expressed genes (DEGs) were identified during leaf development from the young to mature stage in the wild type, while 7,336 DEGs were detected in the mutant. Among these, 5,686 genes exhibited common expression trends in both materials (Fig. 5c). GO enrichment analysis was performed on genes with altered expression patterns during the transition from young to mature leaves in both materials, including response to abscisic acid (ABA), response to ethylene, and response to jasmonic acid (JA). These hormones are well-established as senescence-related, and their signalling pathways were prematurely and strongly activated in the FhATG7 -RNAi lines, potentially triggering a systemic "senescence initiation" signal at the cellular level. This likely represents the most direct molecular switch leading to premature leaf senescence. Notably, in vivo imaging analysis revealed a significant accumulation of ROS in the leaves of the FhATG7 -RNAi line (Fig. 5b), providing an upstream trigger for the premature activation of these hormonal signals. Within this context, alterations in carbohydrate metabolic processes suggested possible dysfunction in photosynthesis, indicating a shift in cellular metabolism from anabolism to catabolism. The upregulation of flavonoid metabolic processes may be viewed as a compensatory protective response aimed at scavenging ROS accumulated due to autophagy deficiency, thereby mitigating oxidative damage. In summary, we propose a mechanism underlying premature senescence: silencing of the FhATG7 gene disrupts autophagic flux, leading to intracellular accumulation of damaged components and ROS, which induces a general cellular stress. This disruption likely prematurely activates senescence-related hormonal signalling pathways, such as ABA and ethylene, during leaf development, ultimately resulting in premature leaf senescence. Discussion Autophagy, a highly conserved cellular degradation and recycling pathway, plays crucial roles in plant growth, development, and stress responses. Although its core machinery is conserved, this study reveals that the regulatory network and physiological contributions of autophagy differ significantly between annual and perennial plants. These differences are primarily reflected in distinct patterns of resource allocation between vegetative and reproductive growth, as well as in long-term stress adaptation strategies. Perennial plants must coordinate growth, reproduction, and stress adaptation across multiple years. Their life-history strategy is a comprehensive trait shaped by resource allocation, source–sink regulation, and other characteristics (Lundgren and Des Marais 2020). By continuously recycling nutrients and removing damaged components, autophagy provides a foundation for perennial plants to maintain meristem activity, achieve periodic organ renewal, and cope with cumulative stresses. This explains why autophagy deficiency leads to more severe premature senescence and growth inhibition in perennial materials. Moreover, autophagy may act through energy sensors such as TOR–SnRK1 to dynamically adjust metabolic strategies under different energy statuses, thereby balancing long-term vegetative growth and reproductive windows(Feng et al 2025). Perennial crops are of great significance for sustainable agriculture due to their well-developed root systems, efficient use of water and nutrients, and carbon sequestration capacity(DeHaan et al 2020; Zhang et al 2023). Future research could integrate autophagy regulation with perennial traits such as root architecture and dormancy characteristics, aiming to develop new crop varieties with short juvenile phases, enhanced stress resistance, and stable yields. This would contribute to the goal of establishing sustainable “plant once, harvest for years” perennial cropping systems. In summary, autophagy serves as a central hub integrating metabolism, development, and stress responses in plants. 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Genome sequencing and CRISPR/Cas9 gene editing of an early flowering Mini‐Citrus (Fortunella hindsii). Plant Biotechnology Journal , 2019, 17:2199-2210 Additional Declarations No competing interests reported. Supplementary Files FigureS1.tif Fig S1. Expression levels of ATG7 in different tissues of Solanum lycopersicum and Fortunella hindsii. a. Expression level of SlATG7 in different tissues of Solanum lycopersicum . b. Expression level of FhATG7 in different tissues of Fortunella hindsii . DPA, days post anthesis; MG, mature green; IMG, immature green; Br, breaker. FigureS2.tif Fig S2. Expression levels of ATG7 in different tissues of Solanum lycopersicum and Fortunella hindsii. a. Phenotype of tomato SlATG7 knockout plants. b. Genotyping of the SlATG7 editing site in tomato revealed that frameshift mutations occurred in all four lines. c. Loss of SlATG7 function leads to late-stage lethality in tomato. FigureS3.tif Fig S3. Phenotypic analysis of fruit morphology and individual fruit weight in SlATG7 RNAi. a. Fruit morphology of SlATG7 -RNAi lines. b. Statistical analysis of individual fruit weight in tomato plants. (n ≥ 20). Data are presented as mean ± SEM. ns, no significant. Scale bar:1cm TableS1.tif Table S1. The number of ATG genes identified in Solanum lycopersicum and Fortunella hindsii. Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-8594705","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":583732432,"identity":"2fafdd54-3138-4075-8ed2-dfd234b77c62","order_by":0,"name":"Guowang Liao","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Guowang","middleName":"","lastName":"Liao","suffix":""},{"id":583732433,"identity":"2f943ac2-8c61-4e93-8b2b-2e4ddea94883","order_by":1,"name":"Haoyang Hu","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Haoyang","middleName":"","lastName":"Hu","suffix":""},{"id":583732434,"identity":"3189bf34-f262-4d47-880b-62e234ecdf4a","order_by":2,"name":"Peng Zhang","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Zhang","suffix":""},{"id":583732435,"identity":"05422dc4-2d9d-4552-a667-4009112557e4","order_by":3,"name":"Ran Hu","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ran","middleName":"","lastName":"Hu","suffix":""},{"id":583732436,"identity":"27e4726a-fe59-47cb-9a20-c3bbf75c5acd","order_by":4,"name":"Saiyu Cao","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Saiyu","middleName":"","lastName":"Cao","suffix":""},{"id":583732437,"identity":"5d3ef2b4-80c3-4e6e-bd8e-cd80f3cf5345","order_by":5,"name":"Ye Guo","email":"","orcid":"","institution":"North West Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Guo","suffix":""},{"id":583732440,"identity":"e8eb2a99-be05-4377-8f83-0f29941dbb72","order_by":6,"name":"Pengwei Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYHADxsbHUJYB0VqajUnVwsAmTZQWg+NnD78uqLhjt+F4c1t1wR+7xAb25m0SDDV3cGs5k5dmPePMs+QNZw623Z7Bk5zYwHOsTILh2DOcWswO5JgZ87YdTja7kdh2m0eCObFBIsdMgrHhMG4t598AtfwDarn/sK2Yx6A+sUH+DQEtN3KMH/M2HLYzu8HYxsyTcBhoCw9+LfY33pgxzzh2OMH+TGKzNM+B48ZtPGnFFgnHcGuR7M8x/lxQc9hesv34w888f6pl+9kPb7zxoQa3FgZodCQ2wLkgIgGfBgYG5s8gB+JXMwpGwSgYBSMaAADrQFkthiLrkQAAAABJRU5ErkJggg==","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Pengwei","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-01-13 17:38:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8594705/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8594705/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101655686,"identity":"d4c05b53-942f-4878-b4bd-f913d5ba7d36","added_by":"auto","created_at":"2026-02-02 09:59:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8247043,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic of Arabidopsis \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eatg7\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and Tomato \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSlATG7\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e RNAi lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Phenotype of 7-day-old \u003cem\u003eArabidopsis \u003c/em\u003eWT and \u003cem\u003eatg7\u003c/em\u003e mutant seedlings grown on 1/2 MS medium. \u003cstrong\u003eb\u003c/strong\u003e. Statistical analysis of root length for Arabidopsis WT and\u003cem\u003e atg7\u003c/em\u003e mutants. (n ≥ 20) \u003cstrong\u003ec\u003c/strong\u003e. Phenotype of Arabidopsis WT and \u003cem\u003eatg7\u003c/em\u003e mutant plants at the rosette stage. \u003cstrong\u003ed\u003c/strong\u003e. Statistical analysis of plant height for Arabidopsis WT and atg7 mutants. (n ≥ 10) \u003cstrong\u003ee\u003c/strong\u003e. Phenotype of tomato WT and \u003cem\u003eSlATG7\u003c/em\u003e-RNAi seedlings. \u003cstrong\u003ef\u003c/strong\u003e. Statistical analysis of hypocotyl length for tomato WT and \u003cem\u003eSlATG7\u003c/em\u003e-RNAi seedlings. \u003cstrong\u003eg\u003c/strong\u003e. Phenotype of 60-day-old tomato WT and \u003cem\u003eSlATG7\u003c/em\u003e-RNAi plants. (n ≥ 10) \u003cstrong\u003eh\u003c/strong\u003e. Statistical analysis of plant height for 60-day-old tomato WT and \u003cem\u003eSlATG7\u003c/em\u003e-RNAi plants. (n ≥ 10). Data are presented as mean ± SEM. Asterisks indicate a significant difference (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 versus the control). Scale bar:1cm (a,e,f) 10cm(c)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8594705/v1/560aa51aa932fcfd6b6e7b99.png"},{"id":101655678,"identity":"29b2706b-0713-4400-bd14-db56c519130b","added_by":"auto","created_at":"2026-02-02 09:59:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7108343,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFortunella hindsii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFhATG7\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-RNAi lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-c\u003c/strong\u003e. Growth phenotypes of one-year-old (a), two-year-old (b), and three-year-old (c) citrus plants （\u003cem\u003eFortunella hindsii\u003c/em\u003e） and the\u003cem\u003e FhATG7\u003c/em\u003e-RNAi lines\u003cem\u003e.\u003c/em\u003e\u003cstrong\u003ed\u003c/strong\u003e. Growth curve showing plant height changes in WT and \u003cem\u003eFhATG7\u003c/em\u003e-RNAi lines over time. Data are presented as mean ± SEM (n ≥ 3). Asterisks indicate a significant difference (***p \u0026lt; 0.001, versus the control). Scale bar:1cm (a) 10cm(b,c)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8594705/v1/686618f9e816b0f04dda69e0.png"},{"id":101753878,"identity":"ddf368c2-68de-4897-9a82-6aa1539800be","added_by":"auto","created_at":"2026-02-03 10:41:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9550052,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic and statistical analysis of reproductive development defects.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Silique phenotype of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e WT and \u003cem\u003eatg7\u003c/em\u003e mutant. \u003cstrong\u003eb\u003c/strong\u003e. Statistical analysis of seed number per silique in WT and \u003cem\u003eatg7\u003c/em\u003e mutant. (n\u0026gt;20) \u003cstrong\u003ec\u003c/strong\u003e. Statistical analysis of seed abortion rate in WT and \u003cem\u003eatg7\u003c/em\u003e mutants. (n\u0026gt;20) \u003cstrong\u003ed\u003c/strong\u003e. Phenotype showing seed number within fruits of tomato WT and \u003cem\u003eSlATG7\u003c/em\u003e-RNAi lines. \u003cstrong\u003ee\u003c/strong\u003e. Statistical analysis of seed number per fruit in tomato \u003cem\u003eSlATG7\u003c/em\u003e-RNAi lines. (n\u0026gt;10) \u003cstrong\u003ef\u003c/strong\u003e. Seed size phenotype of tomato WT and \u003cem\u003eSlATG7\u003c/em\u003e-RNAi lines. \u003cstrong\u003eg\u003c/strong\u003e. Statistical analysis of seed size in tomato WT and \u003cem\u003eSlATG7\u003c/em\u003e-RNAi lines. (n\u0026gt;100) \u003cstrong\u003eh\u003c/strong\u003e. Fruit count per plant of WT and \u003cem\u003eFhATG7\u003c/em\u003e-RNAi lines.\u003cstrong\u003e i\u003c/strong\u003e. Statistical analysis of fruit count in WT and \u003cem\u003eFhATG7\u003c/em\u003e-RNAi lines. (n ≥ 3) \u003cstrong\u003ej\u003c/strong\u003e. Phenotype of abnormal seed development in \u003cem\u003eFhATG7\u003c/em\u003e-RNAi lines. \u003cstrong\u003ek\u003c/strong\u003e. Statistical analysis of flower number in WT and \u003cem\u003eFhATG7\u003c/em\u003e-RNAi lines. (n ≥ 3) Data are presented as mean ± SEM. Asterisks indicate a significant difference (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 versus the control). Scale bar:1cm (a,d,e,f,h,j)\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8594705/v1/b513ef5309b476fa44f73e31.png"},{"id":101753037,"identity":"a7fb226b-3197-4dba-8cd6-cde437cc61b0","added_by":"auto","created_at":"2026-02-03 10:38:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9276036,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic and statistical analysis of reproductive development defects.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Early senescence phenotype of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e \u003cem\u003eatg7\u003c/em\u003e mutant under carbon starvation stress. \u003cstrong\u003eb\u003c/strong\u003e. Measurement of chlorophyll content in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e WT and\u003cem\u003e atg7\u003c/em\u003e mutant seedlings under carbon starvation. \u003cstrong\u003ec\u003c/strong\u003e. Early senescence phenotype of detached leaves from tomato WT and \u003cem\u003eSlATG7\u003c/em\u003e-RNAi lines after dark incubation. \u003cstrong\u003ed\u003c/strong\u003e. Chlorophyll content measurement in tomato WT and \u003cem\u003eSlATG7\u003c/em\u003e-RNAi detached leaves before and after dark incubation. \u003cstrong\u003ee\u003c/strong\u003e. Early senescence phenotype of \u003cem\u003eFortunella hindsii\u003c/em\u003e WT and \u003cem\u003eFhATG7\u003c/em\u003e-RNAi lines. (The numbers 1–6 represent the leaf order from top to bottom along the shoot.) \u003cstrong\u003ef\u003c/strong\u003e. Determination of chlorophyll content in WT and \u003cem\u003eFhATG7\u003c/em\u003e-RNAi leaves. Data are presented as mean ± SEM. Asterisks indicate a significant difference (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 versus the control). Scale bar:1cm (c,e)\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8594705/v1/d1a8a07314462907a703c01f.png"},{"id":101655680,"identity":"8e35f3e0-1b76-44bf-aacb-e9423a0db1bc","added_by":"auto","created_at":"2026-02-02 09:59:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3227512,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome Analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFhATG7\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-RNAi Transgenic Line.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Schematic diagram of transcriptome analysis sampling, showing three biological replicates each of young leaves and mature leaves from both wild-type and \u003cem\u003eFhATG7\u003c/em\u003e RNAi #2 transgenic lines. \u003cstrong\u003eb\u003c/strong\u003e. Representative images of ROS accumulation in the leaves of WT and FhATG7-RNAi plants, stained with the ROS-specific fluorescent dye H₂DCFDA and captured using a live imaging system. \u003cstrong\u003ec\u003c/strong\u003e. Venn diagram of differentially expressed genes (DEGs) during leaf development from young to mature stages in WT and \u003cem\u003eFhATG7\u003c/em\u003eRNAi transgenic lines. \u003cstrong\u003ed\u003c/strong\u003e. GO enrichment analysis of DEGs during leaf development in WT and \u003cem\u003eFhATG7\u003c/em\u003e-RNAi transgenic lines. (2118+1650). Scale bar:1cm (a)\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8594705/v1/4d4273246ef7e3b168d9932f.png"},{"id":101755822,"identity":"ebec0b8c-4fd9-4052-9907-7693369a8c23","added_by":"auto","created_at":"2026-02-03 10:55:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":34519230,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8594705/v1/cd3a37e8-8b95-4422-94c1-ca34cfa3099e.pdf"},{"id":101655683,"identity":"dd167f14-ecd1-4954-a89a-cea0f080f9d3","added_by":"auto","created_at":"2026-02-02 09:59:33","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5652311,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig S1. Expression levels of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eATG7\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in different tissues of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSolanum lycopersicum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFortunella hindsii.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Expression level of \u003cem\u003eSlATG7\u003c/em\u003e in different tissues of \u003cem\u003eSolanum lycopersicum\u003c/em\u003e. \u003cstrong\u003eb\u003c/strong\u003e. Expression level of \u003cem\u003eFhATG7\u003c/em\u003e in different tissues of\u003cem\u003e Fortunella hindsii\u003c/em\u003e. DPA, days post anthesis; MG, mature green; IMG, immature green; Br, breaker.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8594705/v1/be9f00f27b224dcbb99c4d46.tif"},{"id":101655679,"identity":"17f59a0f-bb3e-42c6-b148-bcd8855ddc29","added_by":"auto","created_at":"2026-02-02 09:59:33","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3817303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig S2. Expression levels of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eATG7\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in different tissues of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSolanum lycopersicum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFortunella hindsii.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Phenotype of tomato \u003cem\u003eSlATG7\u003c/em\u003eknockout plants. \u003cstrong\u003eb\u003c/strong\u003e. Genotyping of the \u003cem\u003eSlATG7\u003c/em\u003eediting site in tomato revealed that frameshift mutations occurred in all four lines. \u003cstrong\u003ec\u003c/strong\u003e. Loss of\u003cem\u003e SlATG7\u003c/em\u003efunction leads to late-stage lethality in tomato.\u003c/p\u003e","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8594705/v1/ac300e27d9feffbfb0b0ac47.tif"},{"id":101655681,"identity":"2ff6f05b-fef3-4da1-8fb4-1e405e7f7f56","added_by":"auto","created_at":"2026-02-02 09:59:33","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2375511,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig S3. Phenotypic analysis of fruit morphology and individual fruit weight in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSlATG7\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eRNAi.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Fruit morphology of \u003cem\u003eSlATG7\u003c/em\u003e-RNAi lines. \u003cstrong\u003eb\u003c/strong\u003e. Statistical analysis of individual fruit weight in tomato plants. (n ≥ 20). Data are presented as mean ± SEM. ns, no significant. Scale bar:1cm\u003c/p\u003e","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8594705/v1/bd42620618135a48757801be.tif"},{"id":101655685,"identity":"be3d3469-48d0-409c-a83f-c9f22b427cce","added_by":"auto","created_at":"2026-02-02 09:59:33","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":9732952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1. The number of ATG genes identified in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSolanum lycopersicum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFortunella hindsii.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"TableS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8594705/v1/be56f60526236312e64e1cae.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Autophagy Is More Vital in Tomato and Citrus than in Arabidopsis","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlants exhibit remarkable diversity in strategies to adapt to vastly different environments on Earth. Their life cycles exhibit two distinct patterns: annual plants tend to reproduce once and then die, while perennial plants engage in long-term trade-offs among growth, reproduction, and survival (Lundgren and Des Marais 2020). Therefore, material cycling and energy utilisation within plants are likely different.\u003c/p\u003e\n\u003cp\u003eAutophagy is a highly conserved process in eukaryotes that degrades and recycles cytoplasmic components, including proteins, protein aggregates, and organelles, during development or under environmental stress. In plants, two main types of autophagy have been described: microautophagy and macroautophagy (Ding et al 2018; Petersen et al 2024). In microautophagy, cytoplasmic components are directly engulfed by the vacuole through invagination of the tonoplast. While in macroautophagy, a cup-shaped membrane structure called the phagophore forms around the cargo, expands, and eventually seals into a double-membrane vesicle known as an autophagosome. Subsequently, the outer membrane of the autophagosome fuses with the tonoplast, releasing the autophagic body into the vacuole for degradation (Avin-Wittenberg et al 2018; Marshall and Vierstra 2018). Autophagy is initiated by the ATG1 kinase complex. This complex, comprising the catalytic ATG1 subunit along with regulatory subunits ATG13, ATG11, and ATG101, phosphorylates and activates downstream autophagy components upon its activation (Kamada et al 2000). The phosphatidylinositol 3-kinase (PI3K) complex generates phosphatidylinositol 3-phosphate (PI3P) at the phagophore (Kihara et al 2001), which in turn recruits PI3P-binding proteins such as ATG18 (Proikas-Cezanne et al 2007). Phagophore expansion and maturation depend on lipid delivery mediated by the lipid scramblase ATG9 and the lipid transfer protein ATG2 (Matoba et al 2020; Valverde et al 2019), as well as two ubiquitin-like conjugation cascades that ultimately lead to the attachment of the ubiquitin-like protein ATG8 to phosphatidylethanolamine (PE) on the autophagosome membrane (Ichimura et al 2000). ATG5 and ATG7 are widely recognised as essential regulators of ATG8 lipidation, a landmark process in autophagosome formation and autophagic flux (Ichimura et al 2000; Kim et al 1999; Mizushima et al 1998). In most organisms, interfering with the function of key ATG genes is sufficient to block autophagy activity and induce autophagy-defective phenotypes (Kurusu et al 2014; Tang and Bassham 2018). \u003c/p\u003e\n\u003cp\u003eHowever, while the core regulatory mechanisms of autophagy have been well-characterised in model plants (e.g., \u003cem\u003eArabidopsis thaliana\u003c/em\u003e) and major crops (e.g., rice, \u003cem\u003eOryza sativa\u003c/em\u003e), research on autophagy in horticultural crops remains in its infancy. In this study, we investigated two contrasting horticultural systems: tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) and mini citrus (\u003cem\u003eFortunella hindsii\u003c/em\u003e), as examples for annual and perennial crops, respectively (Zhu et al 2019). We performed comparative phenotype studies using the Arabidopsis \u003cem\u003eatg7\u003c/em\u003e T-DNA knock-out mutant, and the previously generated \u003cem\u003eATG7\u003c/em\u003e-RNAi lines of tomato and citrus (Guo et al 2025a; Guo et al 2025b), and found that autophagy deficiency consistently impairs vegetative growth across all three species. Notably, in the perennial citrus plants (\u003cem\u003eF. hindsii\u003c/em\u003e), the reduction in biomass exhibited a cumulative effect, resulting in progressively more stunted growth over multiple years. Regarding reproductive development, all three autophagy-deficient genotypes exhibited reduced seed production. Under dark-induced carbon starvation, both \u003cem\u003eArabidopsis\u003c/em\u003e \u003cem\u003eatg7\u003c/em\u003e mutants and \u003cem\u003eSlATG7-\u003c/em\u003eRNAi lines exhibited premature leaf senescence. Strikingly, the \u003cem\u003eFhATG7\u003c/em\u003e-RNAi lines displayed a pronounced premature leaf senescence phenotype even under normal growth conditions. To investigate the underlying cause of premature leaf senescence in these autophagy-deficient materials, we measured the reactive oxygen species (ROS) content and found that \u003cem\u003eFhATG7\u003c/em\u003e-RNAi leaves exhibited increased ROS levels. Comparative transcriptome analysis further revealed that knockdown of the \u003cem\u003eFhATG7\u003c/em\u003e gene disrupts autophagic flux, leading to the activation of senescence-related hormonal signalling pathways such as ABA, ethylene, and jasmonic acid, thereby triggering systemic senescence initiation at the cellular level. Collectively, this work elucidates autophagy-dependent developmental regulation in horticultural crops, with particular significance for perennial species, and offers a valuable foundation for targeted breeding strategies to enhance sustainable productivity and stress adaptation in perennial horticultural crops.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll Arabidopsis lines used in this study are of the Columbia-0 (Col-0) background. The \u003cem\u003eatg7\u003c/em\u003e (SAIL_11_H07) mutants have been described previously(Guan et al 2019). The Arabidopsis seeds were surface sterilised and germinated on half Murashige and Skoog (MS) medium containing 0.8% (w/v) agar and 3% (w/v) sucrose, pH 5.8. Seedlings were cultured in a plant growth chamber at 22 \u0026deg;C under a 16-h light and 8-h dark cycle before being transferred into soil in a plant growth room. The \u003cem\u003eFhATG7\u003c/em\u003e-RNAi transgenic citrus (\u003cem\u003eFortunella hindsii\u003c/em\u003e) has been described previously (Guo et al 2025b). The citrus plants were grown in the greenhouse of the Citrus Cultivation Centre at Huazhong Agricultural University (Wuhan, China) under standard cultivation conditions. The \u003cem\u003eSlATG7\u003c/em\u003e-RNAi transgenic tomato (\u003cem\u003eSolanum lycopersicum, \u003c/em\u003eMicro-Tom) has been described previously (Guo et al 2025a). The tomato plants were grown under standard greenhouse conditions (14 h of light at 26\u0026deg;C; 10 h dark at 20\u0026deg;C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of \u003cem\u003eATG\u003c/em\u003e genes in tomato and mini citrus.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHomology searches were performed against the tomato(https://solgenomics.net) and mini\u003c/p\u003e\n\u003cp\u003eCitrus (http://citrus.hzau.edu.cn) genomes using conserved domains from yeast ATG proteins. Simultaneously, amino acid sequences of Arabidopsis ATG proteins were retrieved from the TAIR database (https://www.arabidopsis.org/) and used as queries for BLASTp searches (E‑value \u0026le; 1 \u0026times; 10⁻⁵) in the tomato and mini citrus genomes. Candidate ATG protein sequences were further validated using the NCBI Conserved Domain Database (CDD; https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to confirm the presence of ATG protein domains.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCarbon deprivation treatment. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eArabidopsis seeds were sown on 1/2 MS plates and grown under a 16-h light/8-h dark photoperiod at 22\u0026deg;C. For carbon deprivation treatment, 7-day-old seedlings were transferred onto 1/2 MS plates without sucrose, wrapped in aluminium foil to maintain darkness, and then returned to the same growth chamber for the indicated durations. Seedlings harvested immediately after transfer served as the 0‑day carbon deprivation control (Xue et al 2022). For carbon deprivation of tomato leaves, leaves were excised with scissors, placed in distilled water, and then incubated in darkness for 7 days before sampling.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eChlorophyll content measurement.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh leaf samples (0.2g) were weighed into a 10-mL tube and cut with scissors, then 10 mL of extraction buffer (acetone-ethanol, 1:1, v/v) was added. After 24 h of darkness treatment, the absorbance at 645 and 663 nm was measured using a microplate reader (BioTek Synergy H1), and chlorophyll content was determined using the equation of Lichtenthaler (1987). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROS Visualization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMature leaves from WT and \u003cem\u003eFhATG7\u003c/em\u003e-RNAi lines of \u003cem\u003eFortunella hindsii\u003c/em\u003e were collected. The leaves were incubated in the dark for 30 minutes with 50 \u0026micro;M of the reactive oxygen species (ROS)-specific fluorescent probe H2DCFDA. Subsequently, the leaves were removed from the incubation solution, thoroughly rinsed, and observed using an in vivo imaging system (NightSHADE L985, Berthold) with excitation/emission wavelengths set at 480 nm/520 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal RNA extraction and transcriptome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of four sample groups were selected for this study, encompassing young leaves and mature leaves from both wild-type and \u003cem\u003eFhATG7\u003c/em\u003e-RNAi transgenic lines. Each group consisted of three biological replicates. Total RNA was extracted from leaf tissues using a column-based plant RNA extraction kit. The quality and integrity of RNA samples were assessed using 1.0% agarose gel electrophoresis, a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). For transcriptome sequencing, RNA-seq libraries were prepared using the NEB Next Ultra II RNA Library Prep Kit for Illumina (New England Biolabs Inc., Ipswich, Massachusetts, USA) following the manufacturer\u0026apos;s (Illumina) instructions and were subsequently sequenced on the Illumina Novaseq 6000 platform (Personal Biotechnology Co., Ltd, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential expression gene analysis and GO-term pathway enrichment analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Raw RNA-seq reads were first subjected to quality control using Fastp to remove adapter sequences and low-quality bases. The clean reads were then aligned to the \u003cem\u003eF. hindsii\u003c/em\u003e reference genome using HISAT2. Gene expression levels were quantified via feature Counts and normalised as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Differential expression analysis was performed using the DESeq2 package in R, with genes showing an adjusted *p*-value (FDR) \u0026lt; 0.05 and an absolute log2 fold change \u0026ge; 1 considered significantly differentially expressed. Gene Ontology (GO) enrichment analysis for these significant genes was conducted using the clusterProfiler R package, with terms considered enriched at FDR \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical graphs were performed using GraphPad Prism 9 software. The results were compared using appropriate statistical analysis methods (Tukey\u0026rsquo;s test, one-way ANOVA or two-way ANOVA for multiple comparisons), and data were expressed as the mean \u0026plusmn; SEM. A p-value below 0.05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIdentification and Characterisation of \u003cem\u003eATG\u003c/em\u003e genes in tomato and mini citrus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eATG\u003c/em\u003e genes play crucial roles in autophagy. To investigate the functions of autophagy-related genes in annual and perennial plants, we identified the number of key \u003cem\u003eATG\u003c/em\u003e genes in the genomes of tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) and citrus (\u003cem\u003eFortunella hindsii\u003c/em\u003e) (Table S1). Similar to Arabidopsis, \u003cem\u003eATG1\u003c/em\u003e, \u003cem\u003eATG8\u003c/em\u003e, and \u003cem\u003eATG18\u003c/em\u003e are consistently encoded by small gene families across all three species, whereas the number of other \u003cem\u003eATG\u003c/em\u003e genes varies between species. In Arabidopsis and other annual crops, mutants of ATG genes commonly exhibit characteristic phenotypes such as reduced growth, premature senescence, decreased seed yield, and enhanced sensitivity to abiotic stress (Petersen et al 2024). We selected the \u003cem\u003eATG7\u003c/em\u003e gene as the example and conducted a systematic comparison of autophagy-deficient materials from the perennial plant F. hindsii and from the annual model plant Arabidopsis, as well as from tomato. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Role of Autophagy in Vegetative Growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression pattern indicates that \u003cem\u003eATG7\u003c/em\u003e is essential in both tomato and citrus (Fig. S1), required to maintain basal autophagy and to play essential roles throughout all developmental stages. To generate autophagy-deficient mutants, we knocked out \u003cem\u003eSlATG7\u003c/em\u003e in tomato via CRISPR-Cas9 gene editing. Interestingly, in contrast to the Arabidopsis \u003cem\u003eatg7\u003c/em\u003e loss-of-function mutant, which develops and sets seeds normally, homozygous \u003cem\u003eatg7\u003c/em\u003e knockout in tomato resulted in a lethal phenotype (Fig. S2). These results suggest that autophagy plays differential levels of importance across species. Compared with Arabidopsis, tomato has greater biomass and produces fleshy fruits, which likely impose higher energy demands. We therefore adopted RNA interference (RNAi) and successfully generated transgenic RNAi lines targeting \u003cem\u003eSlATG7\u003c/em\u003e in tomato and \u003cem\u003eFhATG7\u003c/em\u003e in citrus, confirming a significant reduction in autophagy activity in these lines (Guo et al 2025a; Guo et al 2025b).\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, 7-day-old \u003cem\u003eatg7\u003c/em\u003e mutants exhibited slightly shorter roots than the wild type under normal growth conditions (Fig. 1ab). Following transplantation into soil, the inflorescence height was also reduced (Fig. 1cd). These results indicate that autophagy deficiency reduces vegetative growth in Arabidopsis. Correspondingly, \u003cem\u003eSlATG7\u003c/em\u003e-RNAi transgenic tomato lines also displayed pronounced developmental defects during vegetative growth. Hypocotyl length in seedlings of two independent transgenic lines was significantly shorter than that of the wild type, indicating growth inhibition (Fig.1e). Further observation at the flowering stage revealed that the plant height of\u003cem\u003e SlATG7\u003c/em\u003e-RNAi lines was significantly lower than that of the WT (Fig.1fh). These results demonstrate that reduced autophagy levels in tomato significantly inhibit growth.\u003c/p\u003e\n\u003cp\u003eIn citrus, continuous three-year observation of \u003cem\u003eFhATG7\u003c/em\u003e‑RNAi T\u003csub\u003e1\u003c/sub\u003e seedlings revealed that the growth of two transgenic lines was consistently and significantly lower than that of the WT (Fig. 2a\u0026ndash;c). Statistical analysis of plant height indicated that the difference became more pronounced in two‑ and three‑year‑old plants (Fig. 2d). In this perennial species, autophagy deficiency likely leads to reduced plant growth, and such impairment may accumulate over successive growing seasons, resulting in progressively more marked phenotypic divergence.\u003c/p\u003e\n\u003cp\u003eIn summary, autophagy deficiency consistently suppresses vegetative growth in Arabidopsis, tomato, and mini citrus, underscoring the conserved role of autophagy in maintaining basic plant growth capacity. However, the phenotypic severity and temporal pattern of this inhibition exhibit species-specific variation. Complete knockout of \u003cem\u003eATG7\u003c/em\u003e leads to only mild growth defects in Arabidopsis but causes lethality in tomato. In contrast, knockdown of \u003cem\u003eATG7\u003c/em\u003e expression level is sufficient to induce significant growth reduction in both tomato and mini citrus. Furthermore, in perennial citrus plants, autophagy deficiency results in cumulative growth impairment, with phenotypic divergence from the wild type progressively widening over successive years. These findings indicate that the functional importance of autophagy varies across species and that, in perennial plants, autophagy not only regulates growth within a single season but may also play a critical role in interannual resource allocation and long-term survival.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Role of Autophagy in Reproductive Growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn Arabidopsis \u003cem\u003eatg7\u003c/em\u003e mutants, siliques exhibited significantly higher seed abortion rates than in the WT (Fig. 3a-c). In tomato \u003cem\u003eSlATG7\u003c/em\u003e-RNAi lines, no significant differences were observed in overall fruit morphology or individual fruit weight (Fig. S3). However, a marked reduction in the number of seeds per fruit was evident. Furthermore, the seeds exhibited a rough surface and developed brown patches on the seed coat in the dry state, in contrast to the smooth surface and whitish coat colour of WT seeds. Seed size measurements indicated a significant increase in seed length in \u003cem\u003eSlATG7\u003c/em\u003e-RNAi lines (Fig. 3d-g). These results confirm that \u003cem\u003eSlATG7\u003c/em\u003e-RNAi leads to substantial alterations in both seed quantity and morphology. In citrus, \u003cem\u003eFhATG7\u003c/em\u003e-RNAi lines exhibited significantly compromised vegetative growth, leading to a marked reduction in flower production (Fig. 3k). The number of fruits per plant was quantified, revealing a significant decrease in the \u003cem\u003eFhATG7\u003c/em\u003e-RNAi lines compared to the WT control (Fig. 3hi). Seeds of the transgenic lines also displayed obvious developmental abnormalities (Fig. 3j).\u003c/p\u003e\n\u003cp\u003eThese findings suggest that the effect of autophagy on reproductive development is closely tied to the differences between annual and perennial plants. In annual species that invest resources into a single major reproductive event, autophagy deficiency directly impairs the final stage of seed production. In contrast, perennial plants must balance multi‑year cycles of vegetative growth and reproductive allocation. Here, autophagy deficiency primarily reduces overall plant vigour, limiting reproductive potential at the source through decreased flower number, resulting in cumulative declines in reproductive success across seasons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAutophagy in Response to Nutrient Stress in Horticultural Crops\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn higher plants, modulation of nutrient availability\u0026mdash;such as deprivation of carbon or nitrogen sources\u0026mdash;serves as a key regulator of senescence progression (Li and Vierstra 2012; Liu and Bassham 2012). We examined leaf senescence under both nutrient-sufficient and carbon-deficient conditions by measuring chlorophyll content as an indicator of senescence. In Arabidopsis \u003cem\u003eatg7\u003c/em\u003e mutants, no significant difference compared with the wild type was observed under nutrient-sufficient conditions (Fig.4a). However, under carbon-deficient stress, the \u003cem\u003eatg7\u003c/em\u003e mutant exhibited pronounced senescence (Fig.4ab). In tomato, \u003cem\u003eSlATG7\u003c/em\u003e-RNAi lines did not show significant early senescence during the vegetative growth stage (Fig.4c). When tomato leaves were subjected to carbon-deficient stress, leaves of the \u003cem\u003eSlATG7\u003c/em\u003e-RNAi lines turned yellow and chlorophyll content decreased significantly (Fig.4cd). In the perennial mini citrus, the \u003cem\u003eFhATG7\u003c/em\u003e-RNAi lines exhibited obvious premature leaf senescence even under normal growth conditions. We measured chlorophyll content in leaves collected from branches at comparable positions, and the results showed a significant decrease compared with the wild type (Fig. 4ef).\u003c/p\u003e\n\u003cp\u003eThese suggest that the role of autophagy in plant responses to nutrient stress may differ between annual and perennial plants. Annual plants likely rely more heavily on autophagy for rapid resource reallocation during short-term, acute stress to sustain survival. In contrast, perennial plants, due to their long-lived nature, require autophagy to continuously maintain leaf protein homeostasis and nutrient balance under normal conditions, thereby supporting the functional longevity of their perennial organs (e.g., evergreen leaves). Consequently, the disruption of autophagy imposes a more severe homeostatic imbalance in perennial plants, which is sufficient to trigger senescence programs even in the absence of external stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-Seq Analysis of Leaf Transcriptome in \u003cem\u003eFhATG7\u003c/em\u003e-RNAi Lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the causes of premature leaf senescence in the \u003cem\u003eFhATG7\u003c/em\u003e-RNAi line, transcriptome sequencing was performed on young and mature leaves from both the \u003cem\u003eFhATG7\u003c/em\u003e-RNAi line and WT (Fig. 5a). The analysis revealed that 7,804 differentially expressed genes (DEGs) were identified during leaf development from the young to mature stage in the wild type, while 7,336 DEGs were detected in the mutant. Among these, 5,686 genes exhibited common expression trends in both materials (Fig. 5c). GO enrichment analysis was performed on genes with altered expression patterns during the transition from young to mature leaves in both materials, including response to abscisic acid (ABA), response to ethylene, and response to jasmonic acid (JA). These hormones are well-established as senescence-related, and their signalling pathways were prematurely and strongly activated in the \u003cem\u003eFhATG7\u003c/em\u003e-RNAi lines, potentially triggering a systemic \u0026quot;senescence initiation\u0026quot; signal at the cellular level. This likely represents the most direct molecular switch leading to premature leaf senescence.\u003c/p\u003e\n\u003cp\u003eNotably, in vivo imaging analysis revealed a significant accumulation of ROS in the leaves of the \u003cem\u003eFhATG7\u003c/em\u003e-RNAi line (Fig. 5b), providing an upstream trigger for the premature activation of these hormonal signals. Within this context, alterations in carbohydrate metabolic processes suggested possible dysfunction in photosynthesis, indicating a shift in cellular metabolism from anabolism to catabolism. The upregulation of flavonoid metabolic processes may be viewed as a compensatory protective response aimed at scavenging ROS accumulated due to autophagy deficiency, thereby mitigating oxidative damage. In summary, we propose a mechanism underlying premature senescence: silencing of the \u003cem\u003eFhATG7\u003c/em\u003e gene disrupts autophagic flux, leading to intracellular accumulation of damaged components and ROS, which induces a general cellular stress. This disruption likely prematurely activates senescence-related hormonal signalling pathways, such as ABA and ethylene, during leaf development, ultimately resulting in premature leaf senescence.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAutophagy, a highly conserved cellular degradation and recycling pathway, plays crucial roles in plant growth, development, and stress responses. Although its core machinery is conserved, this study reveals that the regulatory network and physiological contributions of autophagy differ significantly between annual and perennial plants. These differences are primarily reflected in distinct patterns of resource allocation between vegetative and reproductive growth, as well as in long-term stress adaptation strategies.\u003c/p\u003e\n\u003cp\u003ePerennial plants must coordinate growth, reproduction, and stress adaptation across multiple years. Their life-history strategy is a comprehensive trait shaped by resource allocation, source\u0026ndash;sink regulation, and other characteristics (Lundgren and Des Marais 2020). By continuously recycling nutrients and removing damaged components, autophagy provides a foundation for perennial plants to maintain meristem activity, achieve periodic organ renewal, and cope with cumulative stresses. This explains why autophagy deficiency leads to more severe premature senescence and growth inhibition in perennial materials. Moreover, autophagy may act through energy sensors such as TOR\u0026ndash;SnRK1 to dynamically adjust metabolic strategies under different energy statuses, thereby balancing long-term vegetative growth and reproductive windows(Feng et al 2025).\u003c/p\u003e\n\u003cp\u003ePerennial crops are of great significance for sustainable agriculture due to their well-developed root systems, efficient use of water and nutrients, and carbon sequestration capacity(DeHaan et al 2020; Zhang et al\u0026nbsp;2023). Future research could integrate autophagy regulation with perennial traits such as root architecture and dormancy characteristics, aiming to develop new crop varieties with short juvenile phases, enhanced stress resistance, and stable yields. This would contribute to the goal of establishing sustainable \u0026ldquo;plant once, harvest for years\u0026rdquo; perennial cropping systems.\u003c/p\u003e\n\u003cp\u003eIn summary, autophagy serves as a central hub integrating metabolism, development, and stress responses in plants. Investigating the specific regulation of autophagy in perennial plants not only expands our understanding of plant life-history strategies but also provides a critical theoretical foundation and molecular toolkit for designing resource-efficient, climate-smart crops for the future.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAvin-Wittenberg T, Balu\u0026scaron;ka F, Bozhkov PV, Elander PH, Fernie AR, Galili G, Hassan A, Hofius D, Isono E, Le Bars R, Masclaux-Daubresse C, Minina EA, Peled-Zehavi H, Coll NS, Sandalio LM, Satiat-Jeunemaitre B, Sirko A, Testillano PS, Batoko H. 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Genome sequencing and CRISPR/Cas9 gene editing of an early flowering Mini‐Citrus (Fortunella hindsii). \u003cem\u003ePlant Biotechnology Journal\u003c/em\u003e, 2019, 17:2199-2210 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Autophagy, Tomato, Citrus, Plant development, Leaf senescence","lastPublishedDoi":"10.21203/rs.3.rs-8594705/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8594705/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Autophagy is crucial for plant growth, development, and stress responses. While its core machinery is conserved across species, the differential regulation of autophagy in annual versus perennial plants, particularly with respect to resource allocation trade-offs, remains poorly understood. Here, we conducted a comparative study to investigate the function of autophagy in horticultural plants with different life cycles. Using autophagy-deficient materials targeting ATG7 in tomato (Solanum lycopersicum) and citrus (Fortunella hindsii), alongside Arabidopsis as a reference, we analysed the impact of autophagy on vegetative growth, reproductive development, and nutrient stress responses in annual and perennial plants. Autophagy deficiency consistently impaired growth across species, but F. hindsii exhibited more severe growth inhibition and premature leaf senescence, highlighting life-history-dependent differences in autophagy reliance. Comparative transcriptomic analysis of F. hindsii further revealed potential molecular networks underlying autophagy deficiency-induced leaf senescence. In conclusion, our study provides the first evidence on how autophagy differentially underpins annual and perennial life strategies, offering a theoretical foundation for future autophagy-based breeding approaches.","manuscriptTitle":"Autophagy Is More Vital in Tomato and Citrus than in Arabidopsis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-02 09:59:28","doi":"10.21203/rs.3.rs-8594705/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"645a1d96-52b6-4d30-b85a-414330a78398","owner":[],"postedDate":"February 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T09:39:01+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-02 09:59:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8594705","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8594705","identity":"rs-8594705","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}