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

Abstract

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.

It is well-accepted that there is a physiological and molecular resemblance between ripening and senescence, as seen in dry fruits such as Arabidopsis siliques (Seymour et al., 2013; Gómez et al., 2014). Indeed, large-scale data analysis suggested that three types of transcriptional circuits controlling ethylene-dependent ripening have evolved from senescence or floral organ identity pathways (Lü et al., 2018). Autophagy is an antisenescence and antiaging mechanism in plants and animals (Liu & Bassham, 2012; Minina et al., 2018; Aman et al., 2021), reconciling with a repressive effect on ripening. We propose that the milder buildup of autophagic capacity in the SlATG-silenced fruits permits earlier and elevated ethylene production, leading to accelerated ripening (Fig. 3h, upper panel). Intriguingly, the participation of autophagy in nonclimacteric fruit ripening of pepper and strawberry was recently reported (López-Vidal et al., 2020; Sánchez-Sevilla et al., 2021). Contrary to our results, it was concluded that autophagy acts in strawberry ripening promotion. If so, we cannot rule out the possibility of autophagy having contradicting roles in climacteric and nonclimacteric fruits. An intriguing possibility is that this reflects the different impacts of ethylene on both fruit types (Perotti et al., 2023). The increased sensitivity of Arabidopsis atg5, atg2, and atg7 to ACC, coupled with their elevated ethylene production (Figs 3a–c, S8), suggests that the repressive effect of autophagy is potentially widespread and may extend to other roles of ethylene beyond ripening, for example during root elongation (Fig. 3h, lower panel). However, we note that fruit ripening and root elongation are independent processes in which identical molecular mechanisms are not necessarily expected to exhibit similar functions. Therefore, the fact that autophagy restricts both of them does not suggest they are linked but rather the outcome of the repressive impact of autophagy on ethylene in both systems. Although the transcript level of genes involved in ethylene production was reportedly elevated in atg5 and atg9 Arabidopsis mutants (Masclaux-Daubresse et al., 2014), we have not detected a similar trend in our ATG4-RNAi L18 fruits (Fig. S6), potentially highlighting the different manner of regulation between both systems.

As it might be difficult to interpret the buildup of autophagy as a repressive mechanism while ripening is ongoing, we wish to clarify this point. Let us assume that ripening progression is determined by the sum of mechanisms that promote and repress it, with the pace determined by their cumulative effects. In other words, such a highly regulated process would require functional breaks from its initiation to control process onset and their full performance at full speed (in our case, at the turning stage) in order to prevent loss of control (i.e. to mediate the transition from climacteric to postclimacteric ethylene production). In a similar manner, autophagy activity increases with leaf senescence progression, although it delays senescence, as is clearly seen in various atg mutants (Yoshimoto et al., 2009). That said, it is not yet fully understood how ripening is initiated. Changes to histone marks and DNA methylation seem to be necessary and are associated with ripening gene activation (Lü et al., 2018; Li et al., 2021). Here, we propose autophagy as an additional layer of regulation.

Several questions emerge from this study. First, how does autophagy limit ethylene? This may happen via the selective degradation of ethylene or ACC production components, such as ACC-Synthase or ACC-Oxidase enzymes (Houben & Van de Poel, 2019; Park et al., 2021). Elimination of even further upstream precursors or other regulatory elements of ethylene production is another possibility. Finally, it cannot be excluded that autophagy may also regulate ethylene signaling components (Binder, 2020). Notably, the ability of ACC to induce autophagy highlights a potential feedback loop for ethylene regulation. Unlike ethylene, ACC levels were reported to continuously increase along with ripening (Van de Poel et al., 2012). Additional questions are as follows what is the weight of ethylene in the well-known early senescence phenotype of autophagy mutants? Especially since, so far, it has mostly been associated with salicylic acid (Yoshimoto et al., 2009). Finally, does autophagy regulate ripening solely via its impact on ethylene? Although the autophagy-ethylene crosstalk seems significant for ripening progression, it is reasonable to assume that several components, such as protein complexes and organelles, not necessarily related to ethylene, would be recycled via selective-autophagy during ripening (Clavel & Dagdas, 2021; Eckardt et al., 2024). Further studies are required to settle these questions.

None declared.

GK, PKP, EQ, S Mursalimov, JD, SA-T, EL and S Michaeli designed experiments and conducted experiments. GK performed all virus-induced gene silencing-related experiments, data and statistical analysis, phylogenetic tree construction and autophagy activity assays in tomatoes. PKP generated the E8:ATG4-RNAi tomato lines and performed most Arabidopsis experiments and SlNBR1 immunoblots. EQ assisted in several aspects of the study and conducted hue, firmness, ethylene measurements and tomato fruit GFP-release assay blots. S Mursalimov performed confocal microscopy and quantification in Arabidopsis. JD examined the E8:ATG4-RNAi lines and performed qPCRs. SA-T assisted in hue, firmness, and ethylene measurements and performed the 1-MCP experiments. EL performed qPCR. JXL and KS generated the GFP-ATG8-2.2 tomato lines. SÜ supervised students and contributed to the draft. All authors discussed the results and contributed to editing the manuscript. S Michaeli conceived the study, supervised the work, and wrote the manuscript. GK and PKP contributed equally to this work.

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