Transcription factor NtWRKY75 accelerates dark-induced leaf senescence through orchestrating hydrogen sulfide homeostasis in Nicotiana tabacum.

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.