S-Nitrosoglutathione works downstream of nitric oxide to mediate iron-deficiency signaling in Arabidopsis

Iron (Fe) is essential for plant growth and development. Knowledge of Fe signaling, from the beginning of perception to activation of the uptake process, is critical for crop improvement. Here, by using chemical screening, we identified a small molecule 3-amino-N-(3-methylphenyl)thieno[2,3-b]pyridine-2-carboxamide named R7 ('R' denoting repressor of IRON-REGULATED TRANSPORTER 1), that modulates Fe homeostasis of Arabidopsis. R7 treatment led to reduced Fe levels in plants, thus causing severe chlorosis under Fe deficiency. Expression analysis of central transcription factors, FER-LIKE IRON DEFICIENCY INDUCED TRANSCRIPTION FACTOR (FIT) and subgroup Ib basic helix-loop-helix (Ib bHLH) genes bHLH38/39/100/101, revealed that R7 targets the FIT-dependent transcriptional pathway. Exogenously supplying S-nitrosoglutathione (GSNO), but not other nitric oxide (NO) donors sodium nitroprusside (SNP) and S-nitroso-N-acetyl-dl-penicillamine (SANP), alleviated the inhibitory effects of R7 on Fe homeostasis. R7 did not inhibit cellular levels of NO or glutathione but decreased GSNO level in roots. We demonstrate that NO is involved in regulating not only the FIT transcriptional network but also the Ib bHLH networks. In addition, GSNO, from S-nitrosylation of glutathione, specifically mediates the Fe-starvation signal to FIT, which is distinct from the NO to Ib bHLH signal. Our work dissects the molecular connection between NO and the Fe-starvation response. We present a new signaling route whereby GSNO acts downstream of NO to trigger the Fe-deficiency response in Arabidopsis.