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 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 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 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 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., 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. 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