Glutamate synthase (GOGAT) is crucial for nitrogen metabolism and amino acid biosynthesis in tea plants, yet the post-transcriptional regulation of GOGAT remains unclear. This study identified miR1507c as a direct interactor of CsNADH-GOGAT, confirmed by DLR assays and 5′RLM-RACE. Notably in tobacco, the relative luciferase activity in plants overexpressing CsNADH-GOGAT and co-expressing miR1507c + CsNADH-GOGATm3 (mutant) were significantly higher than in those co-expressing miR1507c + CsNADH-GOGAT. Overexpression of miR1507c also significantly suppressed the expression of CsNADH-GOGAT and endogenous NtNADH-GOGAT homologs. Leveraging lncRNA sequencing, we screened lncR12304.1 as a ceRNA that regulates CsNADH-GOGAT by competitively binding to miR1507c. Cytoplasmic co-localization (validated by FISH) and direct interaction (confirmed by DLR assays) between lncR12304.1 and miR1507c were established. RNA pull down-qPCR further demonstrated miR1507c binding to both lncR12304.1 and CsNADH-GOGAT. The regulatory axis lncR12304.1–miR1507c–CsNADH-GOGAT was substantiated in vivo: (i) in tea roots/shoots under varying nitrogen treatments and following miR1507c suppression using Antagomir, and (ii) in tobacco via transient co-overexpression. Collectively, our results demonstrate the establishment of this ceRNA network and its role in regulating glutamate and theanine biosynthesis.

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