E3 ligase BRG3 persulfidation delays tomato ripening by reducing ubiquitination of the repressor WRKY71

Abstract

Hydrogen sulfide (H2S) is a gaseous signaling molecule reported to play multiple roles in fruit ripening. However, the molecular mechanisms underlying H2S-mediated delay in fruit ripening remain to be established. Here, the gene encoding a WRKY transcription factor (TF), WRKY71, was identified as substantially upregulated in H2S-treated tomato (Solanum lycopersicum) via transcriptome profiling. Expression of WRKY71 was negatively associated with that of CYANOALANINE SYNTHASE1 (CAS1). Transient and stable genetic modification experiments disclosed that WRKY71 acts as a repressor of the tomato ripening process. CAS1 appears to play an opposite role, based on the finding that the ripening process was delayed in the cas1 mutant and accelerated in CAS1-OE tomatoes. Dual-luciferase reporter assay (DLR), yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and transient transformation experiments showed that WRKY71 bond to the CAS1 promoter and suppressed its activation. Moreover, the persulfidation of WRKY71 enhanced its binding ability to the CAS1 promoter. Data from luciferase complementation and Y2H assays confirmed that WRKY71 interacts with a BOI-related E3 ubiquitin-protein ligase 3 (BRG3) and is ubiquitinated in vitro. Further experiments showed that modification of BRG3 via persulfidation at Cys206 and Cys212 led to reduced ubiquitination activity. Our findings support a model whereby BRG3 undergoes persulfidation at Cys206 and Cys212, leading to reduced ubiquitination activity and decreased interactions with the WRKY71 transcript, with a subsequent increase in binding activity of the persulfidated WRKY71 to the CAS1 promoter, resulting in its transcriptional inhibition and thereby delayed ripening of tomatoes. Our collective findings provide insights into a mechanism of H2S-mediated regulation of tomato fruit ripening.

Persulfidome of Sweet Pepper Fruits during Ripening: The Case Study of Leucine Aminopeptidase That Is Positively Modulated by H2S

“…At present, the most plausible effect of H 2 S at the protein level is the promotion of persulfidation, but this event has been little investigated in fruits and other vegetables. Thus, exogenous H 2 S delayed the ripening process of tomato by persulfidation linked to several proteins, including WRKY6, WRKY71 transcription factor, and E3 ligase BRG3 [ 93 , 94 ], and promoted Arabidopsis flowering by the persulfidation of transcript factor AtU2AF65a [ 50 ]; in pear, the persulfidation of transcription factor MYB10 triggered a lower content of anthocyanin on the skin [ 95 ]. However, to our knowledge, there is no information about any persulfidome analysis of fleshy fruits during their ripening process.…”

Hydrogen Sulfide in the Oxidative Stress Response of Plants: Crosstalk with Reactive Oxygen Species

“…Protein persulfidation, also known as S-sulfhydration, is a reversible process in which RSH is oxidized to a persulfide thiol (RSSH) group. Several reports have indicated that the redox-based reversible post-translational modification not only regulates plant growth and developmental processes but also serves as a protective mechanism for H2S under oxidative stress [40][41][42]. In the following sections, we highlight the molecular mechanisms of persulfidation in the regulation of oxidative stress.…”

Recent Advances in Studying the Regulation of Fruit Ripening in Tomato Using Genetic Engineering Approaches

“…An attempt to understand the hydrogen sulfide-mediated regulation of ripening is made in [291]. The authors suggest that the ubiquitin-protein ligase BRG3 undergoes persulfidation at two cysteine residues, leading to a decrease in ubiquitinating activity and its interaction with the repressor transcription factor WRKY71.…”