Lingxi Geng

Osmotic stress limits rice productivity, yet the crosstalk between hydrogen sulfide signaling and redox regulation remains incompletely understood. We previously showed that redox-dependent oligomerization of the basic (region) leucine zippers transcription factor bZIP68 at Cys245 confers osmotic tolerance. However, the role of an adjacent cysteine, Cys171, was undefined. Here, we demonstrate that osmotic stress induces persulfidation of bZIP68 specifically at Cys171. This modification facilitates Cys245-mediated oxidation-dependent oligomerization, thereby enhancing bZIP68 transcriptional activity toward COLD-REGULATED413-THYLAKOID MEMBRANE1 (COR413-TM1). Transgenic complementation and physiological assays confirmed that Cys171 persulfidation is essential for full bZIP68 function in osmotic adaptation. Transcriptomic analysis further revealed that Cys171 is required for bZIP68-driven transcriptional reprogramming under stress. Our findings establish a hierarchical redox cascade wherein persulfidation primes bZIP68 for oxidative activation, highlighting a regulatory crosstalk between distinct post-translational modifications. These mechanistic insights expand our understanding of H₂S signaling and identify the bZIP68 cysteine network as a potential target for improving crop stress resilience.

The action of abscisic acid (ABA) is closely related to its level in plant tissues. Uridine diphosphate-glycosyltransferase71c5 (UGT71C5) was characterized as a major UGT enzyme to catalyze the formation of the ABA-glucose ester (ABA-GE), a reversible inactive form of free ABA in Arabidopsis thaliana (thale cress). UGTs function in a mode where the catalytic base deprotonates an acceptor to allow a nucleophilic attack at the anomeric center of the donor, achieving the transfer of a glucose moiety. The proteomic data revealed that UGT71C5 can be persulfidated. Herein, an experimental method was employed to detect the persulfidation site of UGT71C5, and the computational methods were further used to identify the yet unknown molecular basis of ABA glycosylation as well as the regulatory role of persulfidation in this process. Our results suggest that the linker and the U-shaped loop are regulatory structural elements: the linker is associated with the binding of uridine diphosphate glucose (UPG) and the U-shaped loop is involved in binding both UPG and ABA.It was also found that it is through tuning the dynamics of the U-shaped loop that is accompanied by the movement of tyrosine (Y388) that the persulfidation of cysteine (C311) leads to the catalytic residue histidine (H16) being in place, preparing for the deprotonation of ABA, and then reorientates UPG and deprotonated ABA closer to the ‘Michaelis’ complex, facilitating the transfer of a glucose moiety. Ultimately, the persulfidation of UGT71C5 is in favor of ABA glycosylation. Our results provide insights into the molecular details of UGT71C5 recognizing substrates and insights concerning persulfidation as a possible mechanism for hydrogen sulfide (H₂S) to modulate the content of ABA, which helps us understand how modulating ABA level strengthens plant tolerance.