Transcription factor <scp>NtWRKY75</scp> accelerates dark‐induced leaf senescence through orchestrating hydrogen sulfide homeostasis in <i>Nicotiana tabacum</i>

As the last stage of leaf development, senescence is orchestrated by an intricate network of endogenous factors and external signals to ensure an efficient recycling of nutrients. Hydrogen sulfide (H₂S) serves as an important gaseous signaling molecule in plants, mediating a myriad of physiological processes like leaf senescence. However, the molecular mechanisms underlying H₂S accumulation and its regulation during leaf senescence in tobacco are still not fully elucidated. In this work, we demonstrate that NtWRKY75, a WRKY transcription factor in tobacco (Nicotiana tabacum), serves as a negative regulator of the expression of the key genes involved in H₂S biosynthesis (l-cysteine desulfhydrase, NtLCD1; d-cysteine desulfhydrase, NtDCD1), thereby accelerating dark-induced leaf senescence. The transcript levels of NtWRKY75 are progressively upregulated during both dark-induced and natural leaf senescence. Transgenic tobacco plants overexpressing NtWRKY75 show premature leaf senescence, while ntwrky75 mutants generated through CRISPR/Cas9 exhibit delayed leaf senescence. Further molecular and biochemical analyses reveal that senescence-associated NtWRKY75 binds to the promoters of NtDCD1 and NtOASA1, downregulating NtDCD1 expression while upregulating NtOASA1, thereby leading to decreased H₂S accumulation. NtWRKY75 also interacts with the promoters of multiple amino acid transporter genes, including NtAAP3, resulting in their upregulation and facilitating amino acid remobilization, which accelerates leaf senescence. Additionally, NtVQ47, a protein containing the VQ motif, physically interacts with NtWRKY75 in vivo and in vitro, thereby fine-tuning its transcriptional activity and influencing leaf senescence. In conclusion, our findings demonstrate that the regulatory network composed of NtVQ47, NtWRKY75, and H₂S plays a crucial role in precisely modulating leaf senescence, offering promising candidates and strategies for future crop improvement.