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Summary: The post‐translational modification of proteins enables cells to respond promptly to dynamic stimuli by controlling protein functions. In higher plants, SPINDLY (SPY) and SECRET AGENT (SEC) are two prominent O‐glycosylation enzymes that have both unique and overlapping roles; however, the effects of their O‐glycosylation on fruit ripening and the underlying mechanisms remain largely unknown. Here we report that SlSPY affects tomato fruit ripening. Using slspy mutants and two SlSPY‐OE lines, we provide biological evidence for the positive role of SlSPY in fruit ripening. We demonstrate that SlSPY regulates fruit ripening by changing the ethylene response in tomato. To further investigate the underlying mechanism, we identify a central regulator of ethylene signalling ETHYLENE INSENSITIVE 2 (EIN2) as a SlSPY interacting protein. SlSPY promotes the stability and nuclear accumulation of SlEIN2. Mass spectrometry analysis further identified that SlEIN2 has two potential sites Ser771 and Thr821 of O‐glycans modifications. Further study shows that SlEIN2 is essential for SlSPY in regulating fruit ripening in tomatoes. Collectively, our findings reveal a novel regulatory function of SlSPY in fruit and provide novel insights into the role of the SlSPY‐SlEIN2 module in tomato fruit ripening. [ABSTRACT FROM AUTHOR]

SUMMARY: Because of a high sensitivity to cold, both the yield and quality of tomato (Solanum lycopersicum L.) are severely restricted by cold stress. The NAC transcription factor (TF) family has been characterized as an important player in plant growth, development, and the stress response, but the role of NAC TFs in cold stress and their interaction with other post‐transcriptional regulators such as microRNAs in cold tolerance remains elusive. Here, we demonstrated that SlNAM3, the predicted target of Sl‐miR164a/b‐5p, improved cold tolerance as indicated by a higher maximum quantum efficiency of photosystem II (Fv/Fm), lower relative electrolyte leakage, and less wilting in SlNAM3‐overexpression plants compared to wild‐type. Further genetic and molecular confirmation revealed that Sl‐miR164a/b‐5p functioned upstream of SlNAM3 by inhibiting the expression of the latter, thus playing a negative role in cold tolerance. Interestingly, this role is partially mediated by an ethylene‐dependent pathway because either Sl‐miR164a/b‐5p silencing or SlNAM3 overexpression improved cold tolerance in the transgenic lines by promoting ethylene production. Moreover, silencing of the ethylene synthesis genes, SlACS1A, SlACS1B, SlACO1, and SlACO4, resulted in a significant decrease in cold tolerance. Further experiments demonstrated that NAM3 activates SlACS1A, SlACS1B, SlACO1, and SlACO4 transcription by directly binding to their promoters. Taken together, the present study identified the miR164a‐NAM3 module conferring cold tolerance in tomato plants via the direct regulation of SlACS1A, SlACS1B, SlACO1, and SlACO4 expression to induce ethylene synthesis. Significance Statement: The present study identified the miR164a‐NAM3 module conferring cold tolerance in tomato plants via the direct regulation of SlACS1A, SlACS1B, SlACO1, and SlACO4 expression to induce ethylene synthesis. [ABSTRACT FROM AUTHOR]