H2S-induced NO/SNO positively promotes betulin production in Betula platyphylla

Abstract

S-nitrosoglutathione reductase (GSNOR) is the pivotal enzyme that mediates cellular levels of nitric oxide (NO) and S-nitrosylation (SNO) in plants. We have previously reported that GSNOR deficiency enhanced betulin production. However, the physiological function of NO/SNO in plants remains unclear, especially for SNO. This study aimed to reveal the role of hydrogen sulfide (H2S)-induced NO/SNO on betulin production in birch (Betula platyphylla). Results showed that the H2S donor sodium hydrosulfide(NaHS)enhanced the production rates of NO, SNO and betulin in birch suspension cells to maximum levels, which was 92.59%, 50.09% and 122.58% higher than that in the control, at 24 h after 1 mmol·L−1 NaHS treatment. NaHS also promoted NO burst, SNO production and the gene expression of farnesyl pyrophosphate synthase, lupeol synthase and squalene synthetase related to betulin synthesis in birch GSNOR transgenic plants by RNA interference silencing. Similarly, pretreatment of the birch suspension cells with the GSNOR inhibitor 3-(5-(4-(1H-imidazol-1-yl) phenyl)-1-(4-carbamoyl-2-methylphenyl)- 1H-pyrrol-2-yl) propionic acid (N6022) promoted NaHS-triggered NO/SNO and betulin production, and the betulin content under NaHS and N6022 treatment was significantly higher than that under NaHS treatment or NaHS and NO specific scavenger 2-(4-carboxyphenyl)-4,4,5,5 -tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). However, pretreatment of the birch suspension cells with cPTIO or NO synthase inhibitor (sodium azide or NG-nitro-L-Arg methyl ester) significantly decreased NaHS-triggered NO/SNO and betulin production. Overall, the above results first verified that endogenous SNO mediated hydrogen sulfide-induced betulin production at the pharmacological and genetic levels, and the homeostasis of NO/SNO mediated hydrogen sulfide-induced betulin production. This study provides new insights into the homeostasis of NO/SNO in plant secondary metabolite production.