Autophagy restricts tomato fruit ripening via a general role in ethylene repression

Ripening involves complex biochemical and molecular reprogramming, resulting in color, texture, aroma, and flavor changes to attract humans and other animals (Giovannoni et al., 2017). In climacteric fruits, this process is controlled by a myriad of phytohormones, predominantly ethylene (Li et al., 2021; Huang et al., 2022). To allow these changes, fruits constantly reshape their cellular proteome by fine-tuning protein degradation and synthesis (Szymanski et al., 2017). While the ubiquitin–proteasome system was shown to be critical in ethylene signaling and ripening (Fenn & Giovannoni, 2021; Jia et al., 2023), knowledge of the impact of autophagy, another central degradation system, is rather limited. Autophagy delivers cytosolic components to the vacuole for degradation and recycling. Double-membrane vesicles, termed autophagosomes, are generated around the cellular cargo destined for degradation. The autophagosome then fuses with the tonoplast to release a single membrane structure, termed autophagic body. Inside of the vacuole, the autophagic body degrades along with its cargo, and its constituents are recycled to replenish cellular energy. Autophagy is executed via the function of > 30 autophagy-related (ATG) proteins (Ding et al., 2018; Marshall & Vierstra, 2018). Notably, ATG proteins annotated with different numbers are not related and have distinct roles in autophagy. Among these, the ubiquitin-like ATG8 proteins are important for autophagosome biogenesis, fusion with the vacuole, and selective recognition of the cargo to be degraded. They are found conjugated either to phosphatidylethanolamine (PE) lipids (lipidated) or in a nonactive (nonlipidated) free form (Kellner et al., 2017). The ATG8 family consists of nine members in Arabidopsis thaliana (Arabidopsis; AtATG8a to AtATG8i). When conjugated to a fluorescent protein, ATG8 family proteins are considered optimal markers for autophagy activity (or flux) assessment, as the stability of the fluorescent protein moiety allows for the estimation of the amount of autophagic material that was delivered to the vacuole. While assessing the ATG8 lipidation status (ATG8-PE : ATG8 ratio) provides a measure of autophagic membrane levels in the cytosol, it does not necessarily reflect autophagy activity (Qi et al., 2023). Due to some level of redundancy (Del Chiaro et al., 2024), ATG8 family members are not used for functional analysis. Alternatively, the downregulation of other essential (and usually single-copy) ATG genes, such as ATG2, ATG5, or ATG7, each independently, can serve for functional analysis (Marshall & Vierstra, 2018). Each of these genes has a critical role in autophagy, rendering the respective knockout mutant autophagy-deficient. ATG2, together with ATG9 and ATG18 family proteins, functions in the lipid delivery system to the developing phagophore. Recently, the ATG2-dependent recruitment of ATG18a onto the phagophore to promote its expansion and closure was revealed (Luo et al., 2023). ATG5 and ATG7 are responsible for different yet essential steps of the ubiquitin-like conjugation of ATG8 proteins to autophagic membranes. ATG4, a protease allowing the lipidation or delipidation of ATG8 members, is represented by a single gene in Solanum lycopersicum (tomato) and two genes in Arabidopsis (AtATG4a and AtATG4b) and is also used for functional analysis (Seo et al., 2016). Initially, it was assumed that ethylene and autophagy are not linked based on the lack of leaf senescence recovery phenotype in Arabidopsis plants deficient in autophagy and Ethylene-Insensitive 2 genes (Yoshimoto et al., 2009). However, several studies later suggested that such an interaction exists (Liao et al., 2022). First, the transcript abundance of several ethylene biosynthesis and ethylene signaling genes was higher in atg5 and atg9 mutants than in wild-type (WT) (Col-0) Arabidopsis plants (Masclaux-Daubresse et al., 2014). Moreover, tomato plants treated with the ethylene precursor, 1-aminocyclopropane-1-carboxylate (ACC), exhibited increased autophagy activity and expression of SlATG8d and SlATG18h genes during drought. The authors suggested the direct binding of Ethylene Response Factor 5 to the promoters of these genes (Zhu et al., 2018). 1-Aminocyclopropane-1-carboxylate was also reported to induce autophagy in Arabidopsis (Rodriguez et al., 2020). Finally, pollination-induced petal leaf senescence, accompanied by enhanced ethylene emission, was further accompanied by increased expression of petunia PhATG8 isoforms and generation of autophagosomes. Notably, the ethylene antagonist, 1-methylcyclopropene (1-MCP), delayed the induction of PhATG8 proteins following pollination and the appearance of the subcellular structures presumed to be autophagosomes (Shibuya et al., 2013). Therefore, ethylene is repeatedly suggested to regulate autophagy. Nonetheless, it is yet unclear whether autophagy has any regulatory impact on ethylene and whether it is involved in climacteric fruit ripening. Tomato (Solanum lycopersicum L.) Micro-Tom, Moneymaker and Ailsa Craig cultivars, and Arabidopsis thaliana (L.) Heynh. Col-0 ecotype were used in this study. Tomatoes were grown in pots at 23°C in a climate-controlled glasshouse under a 12-h, light : dark cycle. Arabidopsis was grown in growth chambers at 16 h : 8 h, 22°C, light : dark cycles. Transgenic Del/Ros1 Micro-Tom (Orzaez et al., 2009) seeds were obtained from Prof. Asaph Aharoni's laboratory (Weizmann Institute). GFP-SlATG8-2.2 Moneymaker lines were generated by transforming the 35S::GFP-StATG8-2.2 construct (Potato StATG8-2.2 is identical to the tomato counterpart), which was kindly provided by Dr Yasin Dagdas (Zess et al., 2019). E8::SlATG4-RNAi lines were generated in a Micro-Tom background. Tomato transformation with the E8::ATG4-RNAi construct was performed using Agrobacterium strain GV3101 harboring the appropriate plasmids as described in Sun et al. (2015) with minor modifications. Tomato seeds were surface-sterilized and germinated on a ½-strength Murashige & Skoog (MS) medium (+30 g l−1 sucrose). After 10 d, fully expanded cotyledons were dissected, with only their central sections used. Explants were immersed in Agrobacterium suspensions (OD600 = 0.6) for 20 min. Explants were then gently dried on Whatman paper and transferred to a cocultivation medium (MS medium supplemented with 3% (w/v) sucrose, 0.8% agar, and 2 mg l−1 6-Benzylaminopurine) for 2 d in the dark. Then, explants were transitioned to a shoot induction medium (MS medium supplemented with 1 mg l−1 zeatin, along with 0.8% (w/v) agar, 3% (w/v) sucrose, 50 mg l−1 kanamycin, and 500 mg l−1 cefotaxime). Incubation took place at 26°C with a 16-h photoperiod. After 30 d, plantlets were separated from the original explants and transferred to a fresh induction medium. Shoots reaching lengths of 2–3 cm were excised and transplanted to a root induction medium (MS medium containing 1 mg l−1 indole-3-acetic acid (IAA), 0.3% (w/v) agar, 3% (w/v) sucrose, and 50 mg l−1 kanamycin). All substances used were from Duchefa Biochemie (Haarlem, the Netherlands). Eventually, 20 lines were obtained, of which five lines (L2, L9, L15, L18, and L20) were examined postharvest, all showing a clear delayed ripening phenotype at their T2 generation. Progeny of some lines, such as L9 (T3 and T4 generations), showed a reduction in phenotype severity, potentially due to trans-generational silencing of the transgene. GFP-SlATG8-2.2 Moneymaker line was generated similarly with a few differences. The seeds were sown on a germination medium (4.3 g l−1 MS including vitamins, 30 g l−1 sucrose, 100 mg l−1 myo-inositol, 0.8% phytoagar, pH 5.8). Two microliters of the Agrobacterium suspension (10 mM MgSO4, 200 μM acetosyringone; OD600 = 1.0) was applied per cotyledon, and they were cocultivated on the germination medium supplemented with 1 mg l−1 6-Benzylaminopurine and 1 mg l−1 NAA for 2 d in the dark at 22°C. In 2 d, the cotyledons were placed abaxial surface down on the germination medium supplemented with 35 mg l−1 kanamycin, 1 mg l−1 trans-Zeatin, and 250 mg l−1 ticarcillin disodium/clavulanate potassium. The cotyledons were placed in 14 h : 10 h, 23°C, light : dark, 50% humidity, and were transferred to a fresh selection medium every 7 d. In the second and fourth weeks, kanamycin concentration was increased to 50 and 100 mg l−1, respectively. Regenerating shoots were cut at the base and transferred to the germination medium supplemented with 20 mg l−1 kanamycin, 0.1 mg l−1 IAA, and vancomycin (500 mg l−1). We employed the ClonExpress II One Step Cloning (Vazyme Biotech, Jiangsu, China) and the Gateway Cloning system (Thermo Fisher Scientific, Waltham, MA, USA) for virus-induced gene silencing (VIGS)-related cloning. The ‘VIGS tool’ at the Sol Genomics Network website (vigs.solgenomics.net) was used to select appropriate 300 base-pair sequences. The primers used are listed in Supporting Information Table S1. Approximately 300-bp fragments of SlATG2 (Solyc01g108160) and SlATG7 (Solyc11g068930) were PCR-amplified and cloned into pENTER-Gus (Thermo Fisher Scientific; modified to have spectinomycin instead of kanamycin resistance). Then, the Gateway LR reaction was performed to allow the transfer of the gene fragment into the pTRV2-Del/Ros1 vector. For ripening-specific SlATG4 (Solyc01g006230) silencing, the E8::SlATG4-RNAi vector was generated by switching the promoter in the 35S::SlATG4-RNAi expression cassette (Alseekh et al., 2022). The original plasmid was amplified (Phusion polymerase) as a linear fragment without the 35S part. Then, ClonExpress II was employed to introduce a PCR-amplified E8 promoter with compatible flanking regions of the linearized plasmid. All cloned plasmids were transformed into the Mix & Go DH5α competent cells (Zymo Research, Irvine, CA, USA) and selected on Luria Broth (LB) agar containing the and plasmids were then using the plasmid tomato fruit were to a using a and One of was with 500 of a The was then to for 5 and at 16 g for 10 min. The resulting was for To allow the of ATG8 and of protein were onto supplemented with in the were onto which were at with 1 : and with : 10 USA) in the MA, USA) were used for : 10 For the protein were onto USA) and with 1 : Arabidopsis were as described et al., 2019). was from 100 mg of or tomato fruits each of using the concentration and were and was from 1 of using the synthesis (Thermo Fisher For reaction the of each was 1 : 10 with with the Mix MA, Then, were using the (Thermo Fisher The was employed for gene expression to genes and and the expression levels and pTRV2-Del/Ros1 harboring the or fragments were into Agrobacterium strain GV3101 using was grown at in a medium containing The the was transferred to a medium containing 10 acid and 20 μM and grown in a Agrobacterium cells were in an medium (10 10 and 200 μM at for h, and to an of Agrobacterium cells were then using a the of fruits to the GFP-SlATG8-2.2 tomato fruits were at different ripening and and in the dark for h and was and using the in system II with an and the Waltham, MA, is as was performed using the system at the sections of tomato fruit or Arabidopsis were a and a containing a medium. for were and were using the or were used. was performed in The were and using For of fruit fruit sections were in supplemented with 2 or 10 μM or the amount of for 7 h or h for For Arabidopsis seeds were germinated in medium containing were transferred into the medium containing either as or 20 μM or 1 μM USA) and or 1 μM and 20 μM were for 2 h in the dark and transferred to in the medium. autophagosomes were from single and cells were in The was as described & with minor modifications. seeds were placed onto containing a medium and sucrose, with or without 20 μM The were then to in for h to light for 2 Then, the were to the growth and in for The of tomato and Arabidopsis ATG8 proteins were from Sol Genomics Network and were using in the using the The resulting was to for which and with was used to the with the the a with was used for were using the and with Finally, the was in the using the of The was and using to and and and the were using the were considered at the Then, fruits were and for and on different postharvest, as in related was using the using a with at a of 1 the in fruits were placed in for 2 Ethylene was by 10 of into a CA, USA) with a and an For of the different were grown on a medium. 1-Aminocyclopropane-1-carboxylate and were applied on by 20 μM in a or to the h of were and in containing a with or without for 2 Each 30 was then with a fresh the into the and into the For fruits from and the E8::ATG4-RNAi lines were placed in a containing of for 20 Then, fruits were and placed in a for the for The leaf from the of E8::ATG4-RNAi lines L15, and and plants was were then and in with Whatman were then with in a growth and at and 14 d. Each were used for each leaf were in 10 of in the dark with at and d using a The was were performed using the from the The was used for > that the are The was used for > two showed and then an was used. a was used. To the role of autophagy in tomato fruit its activity by (Qi et al., 2023). are tomato protein of Arabidopsis into et al., et al., 2019). from the Sol Genomics Network Tomato et al., revealed that five of the members showed expression along ripening Two of these, and exhibited a higher transcript abundance To that as a an protein in fruit generated tomato plants of fruit cells at the in of autophagosomes structures were and to be is a phagophore at an closure by later as a fully and autophagosome To into autophagy protein of tomato fruits at different ripening with an We this all ATG8 analysis showed a in from levels at the to expression at and these levels the The in reflect either an in autophagy activity or a in autophagy to the vacuole. To which of these is GFP-SlATG8-2.2 fruits were to autophagy an in fruits of to However, the was not in any of the fruits examined = We then employed the which allows of autophagy from the to the vacuole to the of the free degradation to the fusion fruit protein of ripening and with revealed a in free level flux) from the to the with analysis the level of at the was than at Nonetheless, it was to a levels of and the free a reduction in autophagy We then examined the impact of a that degradation (Qi et al., 2023). When applied at the and (10 the level of the free degradation that of it was via degradation. further allows of fluorescent proteins the vacuole of following showed structures to autophagic the the vacuole Notably, such autophagic were in any of the following analysis of > 50 cells in 10 different fruits autophagy at the it is at the levels along ripening increased autophagy which at the Autophagy is involved in including and et al., et al., et al., 2017). Therefore, knockout or of any ATG gene in the of ripening-specific To applied two First, used in the Del/Ros1 tomato fruits, harboring the and 1 by the E8 Therefore, silencing which the appearance of fruits (Orzaez et al., is a for silencing of the autophagy genes, SlATG2 or each with or in due to its is with the of in Arabidopsis and mutants et al., et al., 2018; et al., 2023). silencing SlATG2 in of fruit by showed an of fruits to fruits was revealed following silencing of SlATG7 with at an Moreover, fruits exhibited fruit as by For the second to ATG8 members, its expression also along ripening they be to allow lipidation or delipidation of ATG8 proteins For ripening-specific silencing, generated lines harboring a construct (Alseekh et al., under the of a promoter We on line which exhibited silencing, and L9, which was revealed as a line in lipidation and delipidation of ATG8 et al., that autophagy activity via levels not Therefore, to the levels of of 1 a cargo to under autophagy and to reflect the autophagy status & et al., 2022). are two genes in and et al., 2014). silencing plants (Alseekh et al., in the of in the E8::ATG4-RNAi fruits to fruits without a in in transcript levels a autophagic in from and the lines were at the and for fruits ripening and 7 d and L9 fruits Notably, ripening was also in fruits of E8::SlATG4-RNAi lines and these that autophagy is involved in ripening silencing of SlATG4 was reported to in tomato leaf senescence (Alseekh et al., a phenotype of autophagy which has the to fruit and ripening. To that lines phenotype is to fruits, from L9, L15, and L18, as as their showed in leaf senescence of the silencing lines to To the activity of the E8 the expression of the cloned fragment in fruits and of and showed that its expression in fruits on 10 that of the expression in fruits the expression of the the were in expression in the of these that E8 promoter silencing, is to To whether the impact of autophagy on ripening is linked to treated L18, and L9 fruits with the ethylene antagonist, were to were at a of to the ripening the ripening of L18, which exhibited delayed ripening as the fruits that autophagy regulate ripening via interaction with of ethylene showed climacteric and higher ethylene in than in and L9 ethylene 7 d and d L9 However, at d were found to be the due to the usually found fruit around the ripening Notably, fruits ethylene 5 d and d L9 Moreover, fruits not in the expression of tomato or 1 to their levels in fruits genes are involved in climacteric ethylene and fruit (Li et al., that of ripening is not at the level of We further examined ethylene from the fruits with the showed ethylene in the fruits the higher at 14 d revealed a with a ethylene at and 8 d together with the autophagy ripening via its impact on ethylene the of to with ethylene and to such as acid and (Fenn & Giovannoni, To further into the and examined its activity in fruits of the ripening and an ethylene et al., a et al., 2020). In was at the and and d and at potentially an increased of the of the ripening Notably, levels were higher in than in fruits at all the was at any of the fruits examined showed in transcript in to fruits, a autophagy activity in fruits However, that the reduction in expression in fruits does not fully the lack of protein autophagy in a clear that which is in ethylene to which is of ethylene the In other the ethylene and autophagy activity is yet in these To whether autophagy ethylene examined Arabidopsis knockout under the a for or mutants & 2017). the mutants not root growth to Col-0 in the dark. However, were following of and mutants and as a ethylene mutant et al., revealed a for atg5 a root under dark Notably, and exhibited and in ethylene to Col-0 following and ethylene under that the root phenotype of mutants following be due to increased ethylene a potentially role for autophagy in ethylene was reported that autophagy based on numbers and expression of the cargo (Rodriguez et al., 2020). to this with First, ATG8 levels in Arabidopsis Col-0 to or showed that ATG8 and levels were higher in at and h Then, in Arabidopsis to of or revealed an increased of whether was applied or with that autophagosomes and their to the vacuole of using the the showing increased following h of which was with the of To whether function rather than further following ethylene Ethylene autophagy yet at an than 20 μM and We that the in reaching autophagy and ethylene be related to the 2 μM was applied of the 20 μM in an at h was with the by et al. these the of and ethylene to induce autophagy. is that is a and molecular ripening and senescence, as in fruits such as Arabidopsis et al., et al., 2014). analysis suggested that of ripening have from senescence or et al., 2018). Autophagy is an and in plants and animals & et al., 2018; et al., with a on ripening. We that the of autophagic in the fruits and ethylene to ripening the of autophagy in fruit ripening of and was reported et al., et al., to it was that autophagy in ripening the of autophagy roles in climacteric and is that this the different of ethylene on fruit et al., 2023). The increased of Arabidopsis and to with their ethylene that the of autophagy is potentially and to other roles of ethylene for during root However, that fruit ripening and root are in which identical molecular are not necessarily to Therefore, the that autophagy of does not they are linked rather the of the impact of autophagy on ethylene in the transcript level of genes involved in ethylene was in atg5 and atg9 Arabidopsis mutants (Masclaux-Daubresse et al., have not a in fruits potentially the different of it be to the of autophagy as a ripening is to this that ripening is by the of that promote and with the by their In other such a process functional from its to process and their at at the in to of to the from climacteric to ethylene In a autophagy activity with leaf senescence it senescence, as is in mutants (Yoshimoto et al., 2009). it is not yet fully ripening is to and to be and are with ripening gene et al., 2018; et al., autophagy as an of from this study. First, does autophagy via the selective degradation of ethylene or such as or & et al., of further or other regulatory of ethylene is another Finally, it be that autophagy also regulate ethylene signaling components 2020). Notably, the of to induce autophagy a for ethylene levels were reported to along with ripening et al., are as is the of ethylene in the senescence phenotype of autophagy it has with acid (Yoshimoto et al., 2009). Finally, does autophagy regulate ripening via its impact on the for ripening it is to that several such as protein and not necessarily related to be recycled via during ripening & 2021; et al., studies are to these was by from The and the of and and the to and from the to We Dr for the Dr Yasin Dagdas for the 35S::GFP-StATG8-2.2 Prof. Asaph (Weizmann for the Del/Ros1 tomato seeds and the virus-induced gene silencing (VIGS)-related Dr for in the fruit system in Prof. for tomato fruits for some of and Dr for with and wild-type tomato We also the with all are and the of Prof. USA) during a and and performed all virus-induced gene and and autophagy activity in generated the tomato lines and performed Arabidopsis and in several of the and ethylene and tomato fruit performed and in examined the lines and performed in and ethylene and performed the performed and generated the tomato and to the All authors the and to the the the and the and to this with this and All are in the and the Supporting Information Table of or ATG7 virus-induced gene silencing on ATG8 abundance and of the on and ethylene SlATG4 expression along tomato fruit ripening silencing of SlATG4 in E8::ATG4-RNAi fruits ripening to of E8::ATG4-RNAi lines not senescence expression of the fragment used to silencing in the silencing does not in of genes involved in ethylene biosynthesis or of and mutants levels of protein, transcript levels are not atg5 and Arabidopsis increased ethylene and 1-aminocyclopropane-1-carboxylate Ethylene Table used in this study. autophagosomes fruit autophagic of an is not responsible for the or of any Supporting Information by the than be to the The is not responsible for the or of any by the than be to the for the The with to in and in any