SlATG8f enhances tomato thermotolerance and fruit quality via autophagy and HS pathways

SlATG8f enhances tomato thermotolerance and fruit quality via autophagy and HS pathways | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results SlATG8f enhances tomato thermotolerance and fruit quality via autophagy and HS pathways View ORCID Profile Qunmei Cheng , Wen Xu , Cen Wen , Zhuo He , Liu Song doi: https://doi.org/10.1101/2025.09.23.678159 Qunmei Cheng a College of Agriculture, Guizhou University , Guiyang 550000, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Qunmei Cheng Wen Xu a College of Agriculture, Guizhou University , Guiyang 550000, China b Engineering Research Center for Protected Vegetable Crops in Higher Learning Institutions of Guizhou Province , Guiyang 550000, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: 15185145028{at}163.com Cen Wen a College of Agriculture, Guizhou University , Guiyang 550000, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zhuo He a College of Agriculture, Guizhou University , Guiyang 550000, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Liu Song a College of Agriculture, Guizhou University , Guiyang 550000, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Preview PDF Abstract Plant responses to high-temperature stress involve dynamic and complex changes in physiology, biochemistry, and gene expression. Autophagy, a highly conserved intracellular degradation system, facilitates the removal and recycling of damaged cytoplasmic components such as proteins and organelles. It plays an essential role in plant growth, development, and adaptation to various stresses. Although the functions of many autophagy-related genes under high-temperature stress have been characterized, the role of SlATG8f , a key member of the SlATG8 family in tomato, remains poorly understood. In this study, we generated SlATG8f- Overexpressing tomato lines using the recombinant vector pBWA(V)HS. Using quantitative reverse transcription polymerase chain reaction (qRT-PCR), we analyzed physiological indices and expression levels of ATG8 family members and heat shock protein-related genes in fruits of wild-type (WT) and SlATG8f -overexpressing plants across four developmental stages under high-temperature stress. Our results indicate that overexpression of SlATG8f upregulates the expression of autophagy-related and heat shock protein genes, promotes early fruit ripening, and improves fruit quality under high-temperature stress. These findings reveal a regulatory role for SlATG8f in maintaining tomato fruit quality under heat stress and provide a theoretical foundation for tomato variety improvement. Introduction Autophagy is an evolutionarily conserved process in eukaryotes that mediates the recycling of intracellular components and damaged macromolecules, playing a crucial role in plant development and stress adaptation[ 1 ]. Among the core autophagy-related ( ATG) proteins, ATG8 is particularly important for autophagosome formation and function. In plants, the ATG8 gene family has expanded considerably from a single ancestral gene in algae to multiple isoforms in higher plants, reflecting its functional diversification[ 2 ]. Most autophagic receptors and adaptors contain an ATG8 -interacting motif (AIM/UIM), which facilitates specific binding to ATG8 proteins [ 3 ]. Growing evidence indicates that autophagy is activated under various abiotic stresses—such as nutrient deficiency, salinity, drought, and extreme temperatures—where it generally promotes cell survival and helps maintain cellular homeostasis[ 4 ]. However, the role of autophagy can vary depending on the type of stress, tissue, and developmental stage. For example, in barley, autophagy has been linked to programmed cell death in microspores under cold stress, and its inhibition resulted in reduced cell death [ 5 ].suggesting that autophagy can exert either pro-survival or pro-death functions depending on the context [ 6 ]. Heat stress (HS) is a major constraint on crop productivity and quality. It has been shown to induce the expression of autophagy-related genes and promote autophagosome formation [ 7 ]. The extent of autophagic activation depends on stress intensity, duration, and plant genotype. Studies using loss-of-function mutants of Arabidopsis ( ATG5 and ATG7) have revealed increased heat sensitivity, accompanied by more severe damage to photosynthetic and membrane systems[ 8 ]. Conversely, overexpression of certain ATG genes, such as MdATG18a in apple, enhanced thermotolerance by alleviating oxidative damage and protecting chloroplast function[ 9 ]. Several autophagy genes—including ATG5, ATG6, ATG12a, ATG18a, ATG8a, ATG8c, ATG8g , and ATG8i —have been implicated in the plant response to high-temperature stress [ 8 , 10 , 11 ]. However, the function of SlATG8f , a key member of the tomato ATG8 family, under high-temperature conditions remains poorly understood. Tomato ( Solanum lycopersicum L .) is a major greenhouse crop and an important model species for studying fruit development and stress responses. Heat stress (HS) severely impairs multiple reproductive processes, including pollen viability, fertilization, and fruit set, leading to significant yield reduction and quality losses [ 12 , 13 ]. Temperatures exceeding 35 °C disrupt key physiological and biochemical processes, adversely affecting sugar metabolism, pigment accumulation, and fruit firmness [ 14 ].Furthermore, HS alters root–shoot signaling and nutrient partitioning, further compromising fruit development [ 15 ].These detrimental effects underscore the need to elucidate the molecular mechanisms that could be targeted to improve thermotolerance. In this study, we generated SlATG8f- overexpressing transgenic tomato lines and exposed them to continuous high-temperature stress (35 °C) during fruit development. Using quantitative reverse transcription PCR (qRT-PCR), we examined the expression profiles of ATG8 family members and heat shock protein (HSP) genes in both wild-type and transgenic plants across four distinct fruit developmental stages. We also assessed key fruit quality parameters, including sensory properties (external color and firmness), flavor-associated traits (sugar-acid ratio), and health-promoting compounds (carotenoids). Our results indicate that SlATG8f overexpression enhances autophagic activity and upregulates HSP gene expression under heat stress, which promotes earlier fruit ripening and improves overall fruit quality. These findings offer novel insights into the function of SlATG8f in thermotolerance and fruit development, and provide a valuable genetic resource for breeding tomato varieties with enhanced heat resilience and superior fruit quality. Materials and methods Plant material handling and growing conditions In this study,wild-type (WT) and SlATG8f-OE homozygous lines (F2 generation) of ‘Micro-Tom’ tomato were cultivated using a conventional soilless substrate. When the first truss of fruit reached the expansion stage, nine WT and nine transgenic plants were transferred to a climate-controlled growth chamber for heat treatment (35 °Cday/21 °Cnight), with plants grown at 25°Cday /16 °Cnight serving as the control[ 16 ], Fruit samples were collected at four developmental stages: mature green, breaker, orange ripe, and red ripe. Measurements included firmness, soluble solid content, titratable acidity, malondialdehyde (MDA) content, and the activities of key antioxidant enzymes. Additionally, the expression levels of ATG8 family members and heat shock protein genes were analyzed by qRT-PCR. The number of seeds per fruit was recorded, and fruit color progression was monitored throughout ripening. Generation of tomato lines with SlATG8f overexpression The SlATG8f overexpression vector was constructed by cloning the SlATG8f coding sequen ce into the pBWA(V)HS vector. Healthy tomato cotyledons were infected with Agrobacterium t umefaciens strain GV3101 harboring the 35S:: SlATG8f construct. Transgenic lines were generate d through Agrobacterium-mediated transformation. The expression level of SlATG8f in the overe xpression lines (SlATG8f-OE) was confirmed by qRT-PCR. Homozygous F2 plants were selecte d for subsequent experiments [ 17 ] Assessment of fruit color transition and fruit set rate The timing of initial color change (recorded from day 0 of treatment) and the number of fruit set were documented through direct observation across different fruit samples. Analysis of fruit quality parameters and enzyme activities Soluble solids content (SSC) and titratable acidity (TA) were measured using a PAL-BX/ACID1 refractometer (ATAGO, Japan). Carotenoid and malondialdehyde (MDA) levels, as well as activities of relevant enzymes and other physiological indicators, were quantified via UV spectrophotometry.Detailed experimental procedures were performed as described in previously establishedmethods:carotenoids [ 18 ], MDA[ 19 ],antioxidant enzyme activities [ 20 ]. The expression levels of SlATG8f gene and HS gene were detected The expression levels of ATG8 family members and heat shock protein (HSP) response genes were quantified using real-time quantitative polymerase chain reaction (qRT-PCR). Total RNA was extracted from frozen tomato fruit samples with a Tengen RNA extraction kit (DP432, Tengen). Complementary DNA (cDNA) was synthesized using a Tengen reverse transcription kit (KR118), followed by qRT-PCR analysis. Gene-specific primers were designed with Premier 5.0 software and synthesized by Bio Company. The names and sequences of all primers are listed in Table S1. qRT-PCR was performed using the TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) kit (Takara) under the following conditions: each reaction was run in three technical replicates. The tomato Actin gene ( Solyc03g078400) was used as the internal reference, and relative gene expression levels were calculated using the 2 -ΔΔCT method. Data processing and analysis The experiment followed a completely randomized design with independent biological replicates. Each treatment group included three replicates, and random sampling was performed within each group. Data were organized using Microsoft Excel 2019 and analyzed with SPSS Statistics 23.0. Statistical significance was determined using Duncan’s multiple range test, with differences considered significant at P<0.05, indicated by different lowercase letters. Figures were generated using Origin 2022, and all data are presented as mean ± standard deviation. Results High temperature stress improves fruit thermotolerance in SlATG8f-OE plants To evaluate the effect of SlATG8f- overexpression( SlATG8f -OE) on tomato fruit development under heat stress, fruit phenotypes were compared across treatment groups ( Fig. 1 ). Under control conditions, no discernible differences were observed between wild-type (WT) and OE fruits. However, high-temperature stress induced fruit deformation in both genotypes, with more severe malformations evident in OE lines. Anatomical observations indicated that OE fruits accelerated and completed locular gel formation earlier than WT under normal conditions. Under heat stress, gel development was delayed in both WT and OE fruits, although OE plants exhibited greater tolerance compared to WT. Download figure Open in new tab Fig. 1. Fruit phenotypic changes in tomato plants under ambient control and high temperature treatments. WT (Wild Type), OE ( SlATG8f -OE) , Gr(Green ripening),Br(Colour breaking),Or(Orange ripening),Re(Red ripening),scale bar (2 cm). Overexpression of SlATG8f improves fruit set rate under high-temperature stress To evaluate the effect of SlATG8f – overexpression( SlATG8f –OE) on fruit maturation under heat stress, the timing of key developmental stages: Mature green, Breaker, Orange ripe, and Red ripe—was recorded and analyzed ( Fig. 2 ). Under control conditions, all four developmental stages occurred earlier in wild-type (WT) plants than in SlATG8f –OE fruits. In contrast, high-temperature stress delayed maturation in WT plants but promoted it in SlATG8f –OE fruits, resulting in consistently earlier ripening in the transgenic line compared to WT under stress conditions. Download figure Open in new tab Fig. 2. Changes in fruit set in tomato plants under ambient control and high temperature treatments.CK (control), H (high temperature treatment), WT (Wild Type), SlATG8f - OE ( SlATG8f overexpressing plants), (P<0.05) Tomato Fruit Quality under Heat Stress Is Modulated by SlATG8f -OE To evaluate the effect of SlATG8f -OE on tomato fruit quality under high-temperature stress, key quality parameters were measured in wild-type (WT) and SlATG8f -OE fruits under both control and stress conditions ( Fig. 3 ). Download figure Open in new tab Download figure Open in new tab Fig. 3. Changes in fruit quality indexes of tomato plants under ambient control and high temperature treatments.CK (control), H (high temperature treatment), WT (Wild Type), SlATG8f -OE ( SlATG8f overexpressing plants), A (fruit hardness), B (fruit soluble solids), C (titratable acid), D (solids-acid ratio), E (carotenoids content), (P<0.05). Fruit firmness was generally higher in SlATG8f -OE fruits than in WT, except at the green ripe stage, where WT fruits were slightly firmer. High temperature further increased firmness in both genotypes ( Fig. 3A ). Soluble solid content (SSC) was similar between genotypes across treatments; however, heat stress increased SSC at the breaker and red ripe stages while decreasing it at the green and orange ripe stages ( Fig. 3B ). Titratable acid (TA) content was consistently higher in SlATG8f -OE fruits than in WT under all conditions and was slightly enhanced by high temperature across developmental stages ( Fig. 3C ). The solid-acid ratio was higher in WT fruits and was reduced under heat stress in both lines ( Fig. 3D ). Under control conditions, carotenoid content was higher in WT fruits. However, heat stress reduced carotenoid accumulation in WT at the orange and red ripe stages, resulting in lower levels than in OE fruits under stress ( Fig.3E ). Overall, SlATG8f -OE fruits exhibited higher firmness, SSC, and TA under high-temperature stress compared to WT. SlATG8f- Overexpression Modulates Enzyme Activities in Tomato Fruit under High Temperature To assess the effect of SlATG8f -overexpression( SlATG8f -OE) on antioxidant enzyme activities under High-temperature stress, key biochemical indicators were measured in fruits from both wild-type (WT) and SlATG8f - overexpressing ( SlATG8f -OE) plants under control and stress conditions ( Fig. 4 ). Download figure Open in new tab Fig. 4. Determination of antioxidant enzyme activities in tomato fruits under ambient control and high temperature treatments.CK (control), H (high temperature treatment), WT (Wild Type), SlATG8f OE ( SlATG8f overexpressing plants), A (CAT enzyme activity), B (POD enzyme activity), C ((MDA content)) D (SOD enzyme activity), (P<0.05) High-temperature stress increased catalase (CAT) activity, with WT fruits consistently exhibiting higher CAT activity than SlATG8f -OE fruits across all developmental stages ( Fig. 4A ). In contrast, peroxidase (POD) activity was generally elevated in SlATG8f -OE fruits compared to WT. Heat stress enhanced POD activity at the orange and red ripe stages but suppressed it at the green and breaker stages ( Fig. 4B ). Malondialdehyde (MDA) content was higher in WT fruits than in SlATG8f -OE fruits at all stages except orange ripe. Under high temperature, MDA content increased in WT fruits at the orange ripe stage but decreased in the other three stages ( Fig. 4C ). Under control conditions, superoxide dismutase (SOD) activity was higher in SlATG8f -OE fruits than in WT. However, heat stress enhanced SOD activity in WT fruits while reducing it in SlATG8f -OE fruits ( Fig. 4D ). Heat stress modulates the expression of heat-shock protein response genes in Tomato fruit The expression of heat stress response genes was analyzed in wild-type (WT) and SlATG8f -overexpressing ( SlATG8f -OE) fruits under both control and high-temperature conditions ( Fig. 5 ). Undercontrol conditions, SlHSFA2 expression was highest in SlATG8f -OE fruits at the breaker stage and remained stable under heat stress. In contrast, SlHSFA3, SlMYB21, and SlMYB26 sh owed low overall expression, with SlHSFA3 and SlMYB26 l evels higher in WT than in SlATG8f -OE fruits. SlHSFA2 was more highly expressed in WT, whereas SlMYB21 was elevated in SlATG8f -OE fruits. Heat stress upregulated SlHSP20, SlHSP21 , and SlHSP70 in both genotypes, with stronger induction observed in WT. Notably, SlHSP90 expression peaked in WT fruits—at the green ripe stage under control conditions and at the breaker stage under heat stress—and was significantly higher in WT than in SlATG8f -OE fruits across all developmental stages. Download figure Open in new tab Download figure Open in new tab Fig. 5. Differential expression analysis of fruit heat stress protein response genes in tomato plants under ambient control and high temperature treatment. CK (control), H (high temperature treatment), WT (Wild Type), SlATG8f OE ( SlATG8f overexpressing plants), (P<0.05). ATG8 Family Gene Expression in Tomato Fruit under Heat Stress This study aimed to determine whether High-temperature stress enhances autophagic activity in tomato fruit by analyzing the expression of ATG8 family genes via qRT-PCR ( Fig. 6 ). Download figure Open in new tab Download figure Open in new tab Fig. 6. Analysis of differential expression of SlATG8s in fruit of tomato plants under ambient control and high temperature treatments.CK (control), H (high temperature treatment), WT (Wild Type), SlATG8f OE ( SlATG8f overexpressing plants), (P<0.05) The results showed that SlATG8a expression remained stable in both WT and SlATG8f-OE fruits and was not affected by temperature. SlATG8b expression exhibited a unimodal pattern across fruit developmental stages. SlATG8c showed its highest expression in WT fruits under control conditions, with relatively stable expression after the breaker stage. In contrast, SlATG8d expression peaked at the breaker stage in SlATG8f-OE fruits under both conditions, while remaining consistently low in WT. Overall, SlATG8d levels were higher in SlATG8f- OE fruits than in WT, following an initial increase and subsequent decrease. SlATG8e expression was highest in WT fruits at the green ripe stage under control conditions and remained stable thereafter. SlATG8f expression was higher in SlATG8f-OE fruits than in WT at the same stage. Meanwhile, SlATG8g expression was nearly undetectable at the green and breaker stages in WT control fruits and generally displayed an initial increase followed by a decrease across development. Discussion Fruit development is a crucial phase in plant reproduction, involving coordinated processes such as cell division, expansion, and metabolic changes. In tomato, ripening is characterized by color transition, tissue softening, and the accumulation of various metabolites [ 21 , 22 ], Autophagy, a highly conserved degradation pathway in eukaryotes, has been implicated in fruit metabolism and nutrient recycling during development, although its role remains underexplored [ 23 ]. While autophagy has been extensively studied in vegetative tissues across numerous crop species [ 24 ], its function in fruits under abiotic stress conditions is still poorly understood. Emerging evidence indicates that autophagy-related genes are upregulated during late ripening stages in grapes and are essential for quality formation in chili and strawberry fruits [ 25 – 27 ], Consistent with these reports, our findings demonstrate that SlATG8f -OE promotes locular gel liquefaction and enhances tissue integrity under normal conditions. Under high-temperature stress, however, both wild-type (WT) and SlATG8f-OE fruits exhibited morphological deformities and delayed gel development, although the transgenic lines maintained superior firmness, soluble solid content, and titratable acidity. Notably, SlATG8f-OE fruits showed higher carotenoid retention under heat stress compared to WT, which suffered a significant reduction—highlighting the protective role of SlATG8f in mitigating heat-induced impairment of bioactive compounds. This heat sensitivity in carotenoid accumulation aligns with previous observations in tomato under prolonged high-temperature treatment [ 28 ]. Autophagy is recognized as a key response activated under heat stress, facilitating the rem oval of damaged proteins and organelles [ 29 ].Mutations in core autophagy genes such as ATG5 and ATG7 have been shown to increase thermosensitivity in Arabidopsis and tomato [ 8 , 30 ]. highl ighting the importance of autophagy in thermotolerance. The heat stress response involves com plex regulatory mechanisms, including the unfolded protein response and chaperone signaling p athways [ 29 , 31 ]. Our results further demonstrate that overexpression of SlATG8f enhances the e xpression of multiple ATG8 family members and heat shock proteins ( HSPs ) under high-tempera ture conditions. Although certain HSPs, including SlHSP90 , exhibited higher expression in wild-type (WT) fruits, most HSP and autophagy-related genes were significantly upregulated in SlAT G8f -OE fruits. This suggests a coordinated activation of protein quality control pathways media ted by SlATG8f under heat stress. However, there are still some limitations in this study, which also point the way for future in-depth research. Primarily, our conclusions are based on an SlATG8f overexpression model. Although overexpression provides valuable phenotypic insights, the generation and analysis of loss-of-function mutants—for instance, using CRISPR/Cas9—would be essential to fully validate SlATG8f’s role in thermotolerance. Just as [ 32 ] used VIGS technology to demonstrate that SlATG8f coordinates ethylene signaling and chloroplast turnover to drive ripening under normal conditions, obtaining reverse genetic evidence—such as through CRISPR/Cas9 mutagenesis—would unequivocally establish the necessity of SlATG8f in thermotolerance. In addition, [ 33 ]and[ 34 ] confirmed the function of autophagy in aging and stress response using ATG mutants in Arabidopsis thaliana and tomato, respectively, which provides a reference for us to construct stable mutants in the future. Secondly, while this study used qRT-PCR data to suggest elevated autophagy activity, aut ophagy is a dynamic process whose activation is best confirmed through detection of ATG8 lip idation ( ATG8 -PE)—a hallmark of autophagosome formation. Thus, Western blot analysis of lip id-modified SlATG8f protein should be considered the “gold standard” method for verifying enh anced autophagic flux, as strongly recommended in established autophagy monitoring guidelines [ 35 ]. In addition,[ 36 ]Western blot analysis of ATG8 lipidation is widely employed in plant autophagy research. Future studies examining dynamic changes in SlATG8f protein levels and lipidation would substantially strengthen our conclusions. Finally, the mechanism by which SlATG8f regulates HSP gene expression remains unclear. It is unknown whether SlATG8f directly interacts with heat shock transcription factors such as SlHSFA2 or SlHSFA3 , or whether it enhances the heat shock response indirectly through selective autophagy of negative regulators of the HSP pathway. Techniques such as yeast two-hybrid (Y2H), luciferase complementation assay (LCI), or co-immunoprecipitation (Co-IP) could help unravel these interactions. Previous studies offer valuable methodological guidance; for example, the interaction between plant ATG8 and specific target proteins has been well documented[ 37 ] and GmATG8s were found to regulate leaf aging via interaction with GmHSP90 in soybean[ 38 ] These findings provide a useful reference for investigating SlATG8f–HSP interactions in tomato. In conclusion, this study demonstrates that SlATG8f positively contributes to thermotolerance in tomato fruit under high-temperature stress, providing a valuable genetic resource and theoretical foundation for improving crop stress resistance and fruit quality through genetic engineering. Future research should focus on generating stable loss-of-function mutants, direct measurement of autophagic flux at the protein level, and elucidating the interaction between SlATG8f and heat shock signaling components to fully unravel the molecular network mediated by this gene Conclusions In this study, wild-type (WT) and SlATG8f -overexpressing ( SlATG8f -OE) tomato plants were subjected to high-temperature stress (35 °C), with 25 °C serving as the control. Comparative analyses revealed that SlATG8f -OE enhanced thermotolerance during fruit development, as reflected by improved phenotypic traits, accelerated fruit set and color transition, and promoted maturation. Furthermore, SlATG8f -OE elevated the expression of several ATG8 family members and heat shock protein-related genes under high-temperature conditions. These results indicate that SlATG8f overexpression enhances autophagic activity and activates heat stress response pathways, thereby promoting early fruit ripening and improving fruit quality under thermal stress. Our findings underscore the critical role of SlATG8f i n regulating tomato fruit development under abiotic stress and provide a theoretical foundation for improving tomato quality and stress resilience in breeding programs. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper. Author contribution statement Qunmei Cheng: Conceptualization, Writing original draft, Formal analysis, Data curation, Software, Methodology, Validation. Cen Wen: Software, Validation. Zhuo He : Software, Validation. Liu Song: Software, Validation. Wen Xu: Writing - review & editing, Conceptualization, Funding acquisition, Project administration, Resources, Supervision. All authors have read and agreed to the published version of the manuscript. Data availability No data was used for the research described in the article. Funding This work was supported by the National Natural Science Foundation of China (31760594 and 32260754), the Special Project for Cultivating Innovative Talents of Thousand Levels in Guizhou Province[(2018)02], the Guizhou University of Science and Technology Research Start-up Fund for New Faculty [(2016)48]. Acknowledgments The authors are grateful to the National and Local Vegetable Engineering Center (Guizhou) and the Laboratory of the Department of Horticulture (Agricultural College of Guizhou University) for their support to this project. Appendix A. Supplementary data Supplementary Table S1 name primers for each gene and Internal reference gene Actin (Solyc03g078400), Table S2 name qRT-PCR reaction system and reaction program. Footnotes E-mail address: c1533747046{at}163.com ; cenwen1098{at}163.com ; zhuohe0707{at}163.com ; 15085780362{at}163.com ; F-Tel:+86-851-88305271, Fax:+86-851-88305271 Reference 1. ↵ Marshall RS , Vierstra RD . Autophagy:The Master of Bulk and Selective Recycling . Annu Rev Plant Biol . 2018 ; 69 : 173 – 208 . doi: 10.1146/annurev-arplant-042817-040606 PMID: 29539270 OpenUrl CrossRef PubMed 2. ↵ Qi H , Li J , Xia F-N , Chen J-Y , Lei X , Han M-Q , et al. Arabidopsis SINAT Proteins Control Autophagy by Mediating Ubiquitylation and Degradation of ATG13 . Plant Cell . 2020 ; 32 : 263 – 284 . doi: 10.1105/tpc.19.00413 PMID: 31732704 OpenUrl Abstract / FREE Full Text 3. ↵ Birgisdottir Å B , LamarkT , Johansen T. The LIR Motif - Crucial for Selective Autophagy . J Cell Sci 2013 ; 126 : 3237 – 3247 . doi: 10.1242/jcs.126128 PMID: 23908376 OpenUrl Abstract / FREE Full Text 4. ↵ Li W , Chen M , Wang E , Hu L , Hawkesford M-J , Zhong L , et al. Genome-Wide Analysis of Autophagy-Associated Genes in Foxtail Millet (Setaria Italica L.) and Characterization of the Function of SiATG8a in Conferring Tolerance to Nitrogen Starvation in Rice . BMC Genomics 2016 ; 17 : 797 . doi: 10.1186/s12864-016-3113-4 PMID: 27733118 OpenUrl CrossRef PubMed 5. ↵ Bárány I , Berenguer E , Solís M-T , Pérez-Pérez Y , Santamaría M-E , Crespo JL , et al. Autophagy Is Activated and Involved in Cell Death with Participation of Cathepsins during Stress-Induced Microspore Embryogenesis in Barley . Journal of Experimental Botany 2018 ; 69 : 1387 – 1402 . doi: 10.1093/jxb/erx455 PMID: 29309624 OpenUrl CrossRef PubMed 6. ↵ Lenz H-D , Haller E , Melzer E , Kober K , Wurster K , Stahl M , et al. Autophagy Differentially Controls Plant Basal Immunity to Biotrophic and Necrotrophic Pathogens . The Plant Journal 2011 ; 66 : 818 – 830 . doi: 10.1111/j.1365-313X.2011.04546.x PMID: 21332848 OpenUrl CrossRef PubMed Web of Science 7. ↵ Zhai Y , Guo M , Wang H , Lu J , Liu J , Zhang C , et al. Autophagy, a Conserved Mechanism for Protein Degradation, Responds to Heat, and Other Abiotic Stresses in Capsicum Annuum L . Front Plant Sci . 2016 ; 7 : 131 . doi: 10.3389/fpls.2016.00131 PMID: 26904087 OpenUrl CrossRef PubMed 8. ↵ Zhou J , Wang J , Yu J-Q , Chen Z. Role and Regulation of Autophagy in Heat Stress Responses of Tomato Plants . Front. Plant Sci 2014 ; 5 : 174 . doi: 10.3389/fpls.2014.00174 PMID: 24817875 OpenUrl CrossRef PubMed 9. ↵ Huo L , Sun X , Guo Z , Jia X , Che R , Sun Y , et al. MdATG18a Overexpression Improves Basal Thermotolerance in Transgenic Apple by Decreasing Damage to Chloroplasts . Hortic Res 2020 ; 7 : 21 . doi: 10.1038/s41438-020-0243-2 PMID: 32140230 OpenUrl CrossRef PubMed 10. ↵ Fu X-Z , Zhou X , Xu Y-Y , Hui Q-L , Chun C-P , Ling L-L , et al. Comprehensive Analysis of Autophagy-Related Genes in Sweet Orange (Citrus Sinensis) Highlights Their Roles in Response to Abiotic Stresses . Int. J. Mol. Sci 2020 ; 21 : 2699 . doi: 10.3390/ijms21082699 PMID: 32295035 OpenUrl CrossRef PubMed 11. ↵ Jiang Z , Zhu L , Wang Q , HouX. Autophagy-Related 2Regulates Chlorophyll Degradation u nder Abiotic Stress Conditions inArabidopsis. Int. J. Mol. Sci 2020 ; 21 : 4515 . doi: 10.3390/ijms21124515 PMID: 32630439 OpenUrl CrossRef PubMed 12. ↵ Sato S , Peet M-M , Thomas J-F. Physiological Factors Limit Fruit Set of Tomato (Lycopersicon Esculentum Mill.) under Chronic, Mild Heat Stress . Plant Cell & Environment 2000 ; 23 : 719 – 726 . doi: 10.1046/j.1365-3040.2000.00589.x OpenUrl CrossRef 13. ↵ Singh S-P , Pandey T , Srivastava R , Verma P-C , Singh P-K , Tuli R , et al. BECLIN1 from Arabidopsis Thaliana under the Generic Control of Regulated Expression Systems, a Strategy for Developing Male Sterile Plants: BECLIN 1 Regulated Expression for Developing Sterile Plants . Plant Biotechnology Journal 2010 ; 8 : 1005 – 1022 . doi: 10.1111/j.1467-7652.2010.00527.x PMID: 21050365 OpenUrl CrossRef PubMed 14. ↵ Zinn K-E , Tunc-Ozdemir M , Harper J-F. Temperature Stress and Plant Sexual Reproduction: Uncovering the Weakest Links . Journal of Experimental Botany 2010 ; 61 : 1959 – 1968 . doi: 10.1093/jxb/erq053 PMID: 20351019 OpenUrl CrossRef PubMed Web of Science 15. ↵ Merchant , S.S. Rellan-Alvarez R , Lobet G , Dinneny J-R. Environmental Control of Root System Biology . In ANNUAL REVIEW OF PLANT BIOLOGY , VOL 67; Merchant , S.S. , Ed.,, Annual Reviews : Palo Alto 2016 ; 67 : 978-0-8243-0667-0. doi: 10.1146/annurev-arplant-043015-11184 OpenUrl CrossRef 16. ↵ Cappetta E , Andolfo G , Guadagno A , Di Matteo A , Barone A , Frusciante L , et al. Tomato Genomic Prediction for Good Performance under High-Temperature and Identification of Loci Involved in Thermotolerance Response . Hortic Res 2021 ; 8 : 212 . doi: 10.1038/s41438-021-00647-3 PMID: 34593775 OpenUrl CrossRef PubMed 17. ↵ Wen C , Luo T , He Z , Li Y , Yan J , Xu W. Regulation of Tomato Fruit Autophagic Flux and Promotion of Fruit Ripening by the Autophagy-Related Gene SlATG8f . Plants 2023 ; 12 : 3339 . doi: 10.3390/plants12183339 PMID: 37765504 OpenUrl CrossRef PubMed 18. ↵ Cheng Y , Dong Y , Yan H , Ge W , Shen C , Guan J , et al. Effects of 1-MCP on Chlorophyll Degradation Pathway-Associated Genes Expression and Chloroplast Ultrastructure during t he Peel Yellowing of Chinese Pear Fruits in Storage . Food Chemistry 2012 ; 135 : 415 – 422 . doi: 10.1016/j.foodchem.2012.05.017 PMID: 22868108 OpenUrl CrossRef PubMed 19. ↵ Sun M , Liu L. Correlation Analysis of Chlorophyll Content, Malondialdehyde Content, and Alkali Damage Index in Lily Leaves Under Alkali Stress at the Seedling Stage . Northern Horticulture 2018 ; 93 – 98 . DOI: 10.11937/bfyy.20180690 OpenUrl CrossRef 20. ↵ Xu Y. Effects of 1-MCP Treatment on Fruit Quality, Reactive Oxygen Species, and Antho cyanin Biosynthesis Metabolism in’Taoxingli’ Plum from Shengzhou . Master’s Thesis, Zheji ang Gongshang University 2020 . 21. ↵ Ronen G , Cohen M , Zamir D , Hirschberg J. Regulation of Carotenoid Biosynthesis during Tomato Fruit Development: Expression of the Gene for Lycopene Epsilon‐cyclase Is Dow n‐regulated during Ripening and Is Elevated in the Mutant Delta . The Plant Journal 1999 ; 17 : 341 – 351 . doi: 10.1046/j.1365-313X.1999.00381.x PMID: 10205893 OpenUrl CrossRef PubMed Web of Science 22. ↵ Cheniclet C , Rong W-Y , Causse M , Frangne N , Bolling L , Carde J-P , et al. Cell Expansion and Endoreduplication Show a Large Genetic Variability in Pericarp and Contribute Strongly to Tomato Fruit Growth . Plant Physiology 2005 ; 139 : 1984 – 1994 . doi: 10.1104/pp.105.068767 . OpenUrl Abstract / FREE Full Text 23. ↵ Wen C , Luo T , He Z , Li Y , Yan J , Xu W. Regulation of Tomato Fruit Autophagic Flux and Promotion of Fruit Ripening by the Autophagy-Related Gene SlATG8f . Plants 2023 ; 12 : 3339 . doi: 10.3390/plants12183339 PMID: 37765504 OpenUrl CrossRef PubMed 24. ↵ Alseekh S , Zhu F , Vallarino J-G , Sokolowska E-M , Yoshida T , Bergmann S , et al. Autophagy Modulates the Metabolism and Growth of Tomato Fruit during Development . Hortic. Res.-England 2022 ; 9 : uhac129 . doi: 10.1093/hr/uhac129 PMID: 35928403 OpenUrl CrossRef PubMed 25. ↵ Ghan R , Petereit J , Tillett R-L , Schlauch K-A , Toubiana D , Fait A , et al. The Common Transcriptional Subnetworks of the Grape Berry Skin in the Late Stages of Ripening . BMC Plant Biol 2017 ; 17 : 94 , doi: 10.1186/s12870-017-1043-1 PMID: 28558655 OpenUrl CrossRef PubMed 26. López-Vidal O , Olmedilla A , Sandalio L-M , Sevilla F , Jiménez A. Is Autophagy Involved in Pepper Fruit Ripening? Cells 2020 ; 9 : 106 . doi: 10.3390/cells9010106 PMID: 31906273 OpenUrl CrossRef PubMed 27. ↵ Sánchez-Sevilla J-F , Botella M-A ,; Valpuesta V , Sanchez-Vera V. Autophagy Is Required for Strawberry Fruit Ripening . Front. Plant Sci 2021 ; 12 : 688481 . doi: 10.3389/fpls.2021.688481 PMID: 34512686 OpenUrl CrossRef PubMed 28. ↵ Hernández V , Hellín P , Fenoll J , Flores P. Increased Temperature Produces Changes in the Bioactive Composition of Tomato, Depending on Its Developmental Stage . J. Agric. Food Chem 2015 ; 63 : 2378 – 2382 . doi: 10.1021/jf505507h PMID: 25706315 OpenUrl CrossRef PubMed 29. ↵ Yang X , Srivastava R , Howell S-H , Bassham D-C. Activation of Autophagy by Unfolded Proteins during Endoplasmic Reticulum Stress . The Plant Journal 2016 ; 85 : 83 – 95 . doi: 10.1111/tpj.13091 PMID: 26616142 OpenUrl CrossRef PubMed 30. ↵ Zhou J , Wang J , Cheng Y , Chi Y-J , Fan B , Yu J-Q , et al. NBR1-Mediated Selective Autophagy Targets Insoluble Ubiquitinated Protein Aggregates in Plant Stress Responses . PLoS Genet 2013 ; 9 : e1003196 . doi: 10.1371/journal.pgen.1003196 PMID: 23341779 OpenUrl CrossRef PubMed 31. ↵ Liu Y , Burgos J-S , Deng Y , Srivastava R , Howell S-H , Bassham D-C. Degradation of the Endoplasmic Reticulum by Autophagy during Endoplasmic Reticulum Stress in Arabidopsis . Plant Cell 2012 ; 24 : 4635 – 4651 . doi: 10.1105/tpc.112.101535 . OpenUrl Abstract / FREE Full Text 32. ↵ Song L , Xu W , Zha X , Cheng Q , Wu X , Wen C , et al. SlATG8f Coordinates Ethylene Si gnaling and Chloroplast Turnover to Drive Tomato Fruit Ripening . Plant Cell Rep 2025 ; 44 : 200 . doi: 10.1007/s00299-025-03578-8 PMID: 40846794 OpenUrl CrossRef PubMed 33. ↵ 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 . doi: 10.1007/s10495-014-0981-4 PMID: 24682700 OpenUrl CrossRef PubMed 34. ↵ Wang Y , Cai S , Yin L , Shi K , Xia X , Zhou Y , et al. Tomato HsfA1a Plays a Critical Role in Plant Drought Tolerance by Activating ATG Genes and Inducing Autophagy . Autophagy 2015 ; 11 : 2033 – 2047 . doi: 10.1080/15548627.2015.1098798 PMID: 26649940 OpenUrl CrossRef PubMed 35. ↵ Final Draft (Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy (4th Edition).Text) (1).Pdf . doi: https://eprints.whiterose.ac.uk/id/eprint/171641/ 36. ↵ Viret C , Faure M. Lipidation Status of Single Membrane-Associated ATG8 Proteins . Trends in Biochemical Sciences 2021 ; 46 : 787 – 789 . doi: 10.1016/j.tibs.2021.06.004 PMID: 34154877 OpenUrl CrossRef PubMed 37. ↵ Suttangkakul A , Li F , Chung T , Vierstra R-D. The ATG1/ATG13 Protein Kinase Complex Is Both a Regulator and a Target of Autophagic Recycling in Arabidopsis . The Plant Cell 2011 ; 23 : 3761 – 3779 . doi: 10.1105/tpc.111.090993 PMID: 21984698 OpenUrl Abstract / FREE Full Text 38. ↵ Islam Md-M , Ishibash Y , Nakagawa A-C-S , Tomita Y , Iwaya-Inoue M , Arima S , et al. Nitrogen Redistribution and Its Relationship with the Expression of GmATG8c during Seed Filling in Soybean . Journal of Plant Physiology 2016 ; 192 : 71 – 74 . doi: 10.1016/j.jplph.2016.01.007 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted September 25, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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Share SlATG8f enhances tomato thermotolerance and fruit quality via autophagy and HS pathways Qunmei Cheng , Wen Xu , Cen Wen , Zhuo He , Liu Song bioRxiv 2025.09.23.678159; doi: https://doi.org/10.1101/2025.09.23.678159 Share This Article: Copy Citation Tools SlATG8f enhances tomato thermotolerance and fruit quality via autophagy and HS pathways Qunmei Cheng , Wen Xu , Cen Wen , Zhuo He , Liu Song bioRxiv 2025.09.23.678159; doi: https://doi.org/10.1101/2025.09.23.678159 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7643) Biochemistry (17717) Bioengineering (13910) Bioinformatics (42017) Biophysics (21480) Cancer Biology (18628) Cell Biology (25537) Clinical Trials (138) Developmental Biology (13392) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22530) Immunology (17755) Microbiology (40438) Molecular Biology (17200) Neuroscience (88705) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4832) Physiology (7657) Plant Biology (15171) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9828) Zoology (2272)