The m6A reader SlYTH2 negatively regulates tomato fruit aroma by impeding the translation process

Abstract

N⁶-methyladenosine (m⁶A) is a fundamentally important RNA modification for gene regulation, whose function is achieved through m⁶A readers. However, whether and how m⁶A readers play regulatory roles during fruit ripening and quality formation remains unclear. Here, we characterized SlYTH2 as a tomato m⁶A reader protein and profiled the binding sites of SlYTH2 at the transcriptome-wide level. SlYTH2 undergoes liquid-liquid phase separation and promotes RNA-protein condensate formation. The target mRNAs of SlYTH2, namely m⁶A-modified SlHPL and SlCCD1B associated with volatile synthesis, are enriched in SlYTH2-induced condensates. Through polysome profiling assays and proteomic analysis, we demonstrate that knockout of SlYTH2 expedites the translation process of SlHPL and SlCCD1B, resulting in augmented production of aroma-associated volatiles. This aroma enrichment significantly increased consumer preferences for CRISPR-edited fruit over wild type. These findings shed light on the underlying mechanisms of m⁶A in plant RNA metabolism and provided a promising strategy to generate fruits that are more attractive to consumers.

Keywords: RNA methylation; flavor quality; phase separation; translation repression.

Fig. 1.

SlYTH2 is essential for aroma-associated volatiles synthesis in tomato fruits. (A) Tomato fruit ripening process of WT and slyth2 mutants. Br, breaker; B + n, n days after breaker. (Scale bar, 1 cm.) (B) Fruit firmness of WT and slyth2 mutants during fruit ripening. Data are presented as mean ± SD of nine independent biological replicates, each indicated by one dot. Different lowercase letters indicate significant differences (Tukey’s multiple range test, P < 0.05). (C) E-nose with principal component analysis of WT, slyth2 #1, and slyth2 #2 fruits at B + 7 stage. Each cluster consists of six replicates (three biological replicates × two technical replicates), each replicate represented by one dot. (D) Content of total flavor-related volatiles during WT and slyth2 mutant fruit ripening. (E) Major classes of fruit volatiles in WT and slyth2 mutant fruits at B + 7 stage. Data in (D) and (E) are indicated as mean ± SD of three biological replicates, each dot represents one replicate. Significant differences (Tukey’s multiple range test, P < 0.05) are denoted with different lowercases. (F) Aroma evaluations by untrained panelists (n = 28) and preference tests (n = 28) of WT and slyth2 mutant fruits at B + 7 stage.

Fig. 2.

The m⁶A binding of SlYTH2 to volatile synthesis-related genes SlHPL and SlCCD1B is associated with the formation of fruit aroma. (A) in vivo FA-RIP-LC–MS/MS showing that m⁶A is enriched in SlYTH2-Flag-IP sample compared with input and IgG-IP samples. (B) Venn diagrams showing the overlap of identified SlYTH2-binding peaks and m⁶A peaks corresponding to 3,850 transcripts (termed SlYTH2 & m⁶A targets). (C) Distribution of SlYTH2 & m⁶A targeted sites across transcript with three nonoverlapping segments (5′ UTR, CDS, and 3′ UTR). UTR, untranslated region; CDS, coding sequence. (D) Sequence motif identified within SlYTH2 & m⁶A targeted sites by HOMER software. (E) Integrative Genomics Viewer (IGV) showing the distributions of SlYTH2-targeted peaks and m⁶A peaks in transcripts related to volatile synthesis: SlHPL and SlCCD1B. The light-yellow rectangle indicates the position of SlYTH2 & m⁶A targeted sites. The input and IgG-IP reads are presented in the foreground. (F) m⁶A-IP-qPCR showing the enrichment of SlHPL and SlCCD1B in m⁶A-IP sample compared to Input. (G) RIP-qPCR showing the binding affinity of SlYTH2 to SlHPL and SlCCD1B in vivo. Data in (A), (F), and (G) are shown as mean ± SD (n = 3, each biological replicate is marked with one dot). ** indicates significant difference of P < 0.01 with unpaired t test.

Fig. 3.

SlYTH2 inhibits translation efficiency of SlHPL and SlCCD1B, thereby suppressing protein accumulation. (A) Quantitative sucrose density gradient analysis showing the polysome profiles of WT and slyth2 #1 fruits. (B) Translation efficiency of SlHPL and SlCCD1B in WT and slyth2 #1 fruits. The translation efficiency of each fraction was measured by the abundance ratio of RNAs in corresponding recovered fractions versus the total RNAs. Data are shown as mean ± SD (n = 3, each biological replicate is marked with one dot). NS, not significant, **P < 0.01 with Sidak’s multiple comparisons test. (C) Venn diagrams showing the overlap of the 4,137 proteins detected in WT and slyth2 #1 proteomes with SlYTH2 & m⁶A targets. (D) Violin plot showing relative protein levels of the 1,649 overlapped proteins obtained in (C) in WT and slyth2 #1 fruits. **P < 0.01, unpaired t test. (E) Relative protein levels of SlHPL and SlCCD1B in WT and slyth2 #1 fruits. Data are shown as mean ± SD (n = 3, each biological replicate is marked with one dot). *P < 0.05, unpaired t test. (F) Schematic diagram of the pCAMBIA1300 vector containing CDS and 3′UTR fragment of SlHPL and SlCCD1B genes. (G) Protein blot assay showing the accumulation of SlHPL-Flag and SlCCD1B-Flag is inhibited by SlYTH2-Myc protein in tobacco leaves. Actin was used as a loading control. Two lanes indicate two replicates.

Fig. 4.

SlYTH2 undergoes phase separation and sequesters target m⁶A–RNAs into condensates. (A) PrLD and IDR domains in SlYTH2 were predicted using “Prion-Like Amino Acid Composition” (PLAAC; http://plaac.wi.mit.edu/) and “Predictor of Natural Disordered Regions” (PONDR; http://pondr.com/), respectively. (B) Visualization of turbid solution of GST-SlYTH2 proteins under PEG8000 treatment. GST and Bovine serum albumin (BSA) proteins serve as controls. (C) Fluorescence image showing in vitro LLPS of recombinant GFP-tagged SlYTH2 proteins (GFP-SlYTH2). (Scale bar, 20 μm.) GFP and GFP-SlYTH2 proteins in droplet-promoting buffer containing 10% PEG8000. The inner box indicates the enlarged area (Scale bar, 5 μm). (D) Time-lapse microscopy showing dynamic fusion of GFP-SlYTH2 proteins. (Scale bar, 2 μm.) (E) FRAP assay and quantification data showing the dynamic property of GFP-SlYTH2 droplets. (Scale bar, 2 μm.) Data are presented for three independent FRAP events. (F) Fluorescence image showing condensate formation of GFP-SlYTH2 with Cy3-labeled m⁶A-modified and Cy5-lebeled non-m⁶A-modified RNA probes of SlHPL and SlCCD1B, respectively. Green, GFP-SlYTH2; red, Cy3-labeled probe; blue, Cy5-labeled probe; yellow, merged images. (Scale bar, 5 μm.) (G) Colocalization intensity of GFP-SlYTH2 with Cy3-labeled m⁶A-modified RNA probes or Cy5-labeled non-m⁶A-modified RNA probes of SlHPL and SlCCD1B, respectively. The colocalization intensity was quantified using 10 different confocal images. Data are presented as mean (n = 10, each dot represents one replicate). **P < 0.01 with unpaired t test.

Fig. 5.

SlYTH2 forms cytosolic condensates with translational regulators. (A) Scatterplot showing SlYTH2-interacting proteins in tomato fruits. The dashed line indicates the threshold of significantly enriched proteins (FC > 4 and P < 0.01). (B) GO enrichment analysis showing protein partners of SlYTH2 in (A) are significantly enriched in RNA binding, peptide metabolic and translation related pathways. (C) BiFC assays showing that SlYTH2 interacts with translational regulators and forms cytosolic condensates, respectively. N. benthamiana leaves were used for transient coexpression and confocal observation. (Scale bar, 20 μm.) (D) Model for the m⁶A reader SlYTH2 in regulation of tomato fruit aroma. SlYTH2, an m⁶A reader, selectively binds to m⁶A-modified transcripts associated with fruit volatile synthesis and promotes LLPS into liquid-like condensates with translational regulators. This interaction impedes ribosome occupancy and translation of target mRNAs, thereby regulating the synthesis of fruit aroma.