SPINDLY interacts with EIN2 to facilitate ethylene signalling-mediated fruit ripening in tomato.

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.

Amyotrophic lateral sclerosis (ALS) linked mutation in Ubiquilin 2 affects stress granule assembly via TIA-1.

AIMS: The ubiquilin-like protein ubiquilin 2 (UBQLN2) is associated with amyotrophic lateral sclerosis and frontotemporal degeneration (ALS/FTD). The biological function of UBQLN2 has previously been shown to be related to stress granules (SGs). In this study, we aimed to clarify the regulatory relationship between UBQLN2 and SGs. METHODS: In this study, we transfected UBQLN2-WT or UBQLN2-P497H plasmids into cell lines (HEK293T, HeLa), and observed the process of SG dynamics by immunofluorescence. Meanwhile, immunoblot analyses the protein changes of stress granules related components. RESULTS: We observed that ubiquilin 2 colocalizes with the SG component proteins G3BP1, TIA-1, ATXN2, and PABPC1. In cells expressing WT UBQLN2 or P497H mutants, in the early stages of SG formation under oxidative stress, the percentage of cells with SGs and the number of SGs per cell decreased to varying degrees. Between WT and mutant, there was no significant difference in eIF2α activity after stress treatment. Interestingly, the UBQLN2 P497H mutant downregulates the level of TIA-1. In addition, the overexpression of the UBQLN2 P497H mutant inhibited the phosphorylation of 4E-BP1 and affected the nucleoplasmic distribution of TDP-43. CONCLUSIONS: Ubiquilin 2 colocalizes with the SG component proteins G3BP1, TIA-1, ATXN2, and PABPC1. It participates in regulating SG dynamics. And UBQLN2 mutation affects the assembly of stress granules by regulating TIA-1. In addition, the overexpression of the UBQLN2 P497H mutant inhibited the phosphorylation of 4E-BP1 and affected the nuclear and cytoplasmic distribution of TDP-43. These provide new insights into the role of UBQLN2 in oxidative stress and the pathogenesis of ALS.