Regulation of Tomato Fruit Autophagic Flux and Promotion of Fruit Ripening by the Autophagy-Related Gene SlATG8f

Abstract

Autophagy is a highly conserved self-degradation process that involves the degradation and recycling of cellular components and organelles. Although the involvement of autophagy in metabolic changes during fruit ripening has been preliminarily demonstrated, the variations in autophagic flux and specific functional roles in tomato fruit ripening remain to be elucidated. In this study, we analyzed the variations in autophagic flux during tomato fruit ripening. The results revealed differential expression of the SlATG8 family members during tomato fruit ripening. Transmission electron microscopy observations and dansylcadaverine (MDC) staining confirmed the presence of autophagy at the cellular level in tomato fruits. Furthermore, the overexpression of SlATG8f induced the formation of autophagosomes, increased autophagic flux within tomato fruits, and effectively enhanced the expression of ATG8 proteins during the color-transition phase of fruit ripening, thus promoting tomato fruit maturation. SlATG8f overexpression also led to the accumulation of vitamin C (VC) and soluble solids while reducing acidity in the fruit. Collectively, our findings highlight the pivotal role of SlATG8f in enhancing tomato fruit ripening, providing insights into the mechanistic involvement of autophagy in this process. This research contributes to a better understanding of the key factors that regulate tomato fruit quality and offers a theoretical basis for tomato variety improvement.

Keywords: SlATG8f; autophagic flux; autophagosome; fruit ripening; tomato.

Figure 1

Differential Expression Analysis of SlATG8s During Tomato Fruit Ripening. The expression profiles of SlATG8s during different stages of tomato fruit ripening are shown. The stages of fruit ripening are as follows: green ripe (Day 30), breaker (Day 35), orange ripe (Day 40), and red ripe (Day 45). Abbreviations: Gr—Green Ripe; Br—Breaker; Or—Orange Ripe; Re—Red Ripe. (A–G)—SlATG8a, SlATG8a, SlATG8b, SlATG8c, SlATG8d, SlATG8e, SlATG8f, SlATG8g genes relative expression. The presented data represent the mean values, and error bars represent the standard deviation. The same letter indicates no significant difference in mean values at p < 0.05 (Duncan’s test).

Figure 2

Identification of SlATG8f OE Plants Line and Phenotypic Observation of Fruits. (A) Quantitative real-time PCR (qRT-PCR) analysis of SlATG8f gene expression in the SlATG8f OE plants line compared to the wild-type (WT) plants. (B) Phenotypic observations and cross-sections at 10 days, 15 days, 20 days, 30 days, 35 days, 40 days, and 45 days after flowering. Abbreviations: WT—Wild Type; SlATG8f-OE—SlATG8f overexpressing plants. The data shown are the mean values of three replicates, and the error bars represent the standard deviation. The same letter indicates no significant difference in mean values at p < 0.05 (Duncan’s test).

Figure 3

Effects of SlATG8f OE Plants on Autophagic Activity in Tomato Fruits. (A) Autophagosomes stained with MDC (green) in SlATG8f OE plants’ and WT fruits during different stages: green ripe (Gr), breaker (Br), orange ripe (Or), and red ripe (Re). Scale bars = 20 μm. (B) Relative autophagic activity quantified by counting the number of autophagosomes stained with MDC in each image from Panel A. (C) Western blot analysis of ATG8 protein expression in SlATG8f OE plants’ and WT fruits at different stages using Actin as a loading control. (D) Quantification of the intensity of each band from Panel C normalized to the intensity of the Actin band. ATG8/Actin ratio represents the relative expression of ATG8 protein. Abbreviations: white scale—20 μm; Gr—Green Ripe; Br—Breaker; Or—Orange Ripe; Re—Red Ripe; WT—Wild Type; SlATG8f-OE—SlATG8f overexpressing plants. The data shown are the mean values of three replicates, and the error bars represent the standard deviation. The same letter indicates no significant difference in mean values at p < 0.05 (Duncan’s test).

Figure 4

Effects of SlATG8f OE Plants on Autophagic Activity in Tomato Fruits. (A1–D2) Autophagic structures in WT tomato fruits at four stages of ripening. (E1–H2) Autophagic structures in SlATG8f OE plants’ tomato fruits at four stages of ripening. Scale bars = 2 μm. WT—Wild Type; SlATG8f-OE—SlATG8f overexpressing plants. Abbreviations: red scale—2 μm; Red arrows indicate autolysosomes (single-membrane structures with cytoplasmic components degraded), and black arrows indicate autophagosomes (double or multi-membrane vesicular structures containing cytoplasmic components). Gr—Green Ripe; Br—Breaker; Or—Orange Ripe; Re—Red Ripe; WT—Wild Type.

Figure 5

Effects of SlATG8f OE Plants on the Expression of Ethylene-Related Genes in Tomato Fruits. (A) qRT-PCR analysis of the expression of the ethylene signal transduction gene ERF2. (B–E) qRT-PCR analysis of the expression of ethylene receptor genes NR, ETR1, ETR4, and ETR3. (F–H) qRT-PCR analysis of the expression of ethylene synthesis genes ACC4, ACO1, and ACC2. Annotations: Gr—Green Ripe; Br—Breaker; Or—Orange Ripe; Re—Red Ripe; WT—Wild Type; SlATG8f-OE—SlATG8f overexpressing plants.The data shown are the mean values of three replicates, and the error bars represent the standard deviation. The same letter indicates no significant difference in mean values at p < 0.05 (Duncan’s test).

Figure 6

Effects of SlATG8f OE Plants on Pigment Accumulation, Soluble Solids, and Acidity in Tomato Fruits. (A) Ascorbic acid (AsA) content in tomato fruits. (B) Soluble solids (SSC) content in tomato fruits (expressed as a percentage). (C) Titratable acidity (TA) content in tomato fruits (expressed as a percentage). (D) Lycopene content in tomato fruits. (E,F) qRT-PCR analysis of the expression levels of lycopene synthesis genes SlPSY1 and SlCRTISO. qRT-PCR primer sequences are provided in Supplementary Table S1. Annotations: Gr—Green Ripe; Br—Breaker; Or—Orange Ripe; Re—Red Ripe; WT—Wild Type; SlATG8f-OE—SlATG8f overexpressing plants. The data shown are the mean values of three replicates, and the error bars represent the standard deviation. The same letter indicates no significant difference in mean values at p < 0.05 (Duncan’s test).