Mild osmotic stress promotes 4-methoxy indolyl-3-methyl glucosinolate biosynthesis mediated by the MKK9-MPK3/MPK6 cascade in Arabidopsis

MKK9-MPK3/MPK6 cascade positively regulates IGSs' biosynthetic genes. Glucosinolates (GSs), secondary metabolites well known for their roles in plant defense, have been implicated to play an important role in plant abiotic stress response; however, the exact role in these processes and the underlying regulatory mechanisms remain elusive. Mitogen-activated protein kinase (MAPK) cascades are extensively involved in plant abiotic stress response. In this study, we examined the levels of four indolic glucosinolates (IGSs) in the shoots of Arabidopsis seedlings under mild osmotic stress conditions and found that 4-methoxy indolyl-3-methyl glucosinolate (4MI3G) accumulated and that MPK3 and MPK6 were activated. Loss of MPK3 or MPK6 function led to a reduction in mild osmotic stress-induced 4MI3G. Further analyses revealed that MKK9 acts upstream of MPK3 and MPK6 to promote 4MI3G accumulation. The level of 4MI3G induced by mild osmotic stress was reduced in the mkk9 mutant. Conversely, 4MI3G increased in MKK9 (DD) , a constitutively activate mutant of MKK9. Gene expression analyses indicated that the activated MKK9-MPK3/MPK6 cascade upregulates the IGS biosynthetic genes. Moreover, the lack of MYB51, the transcription factor controlling biosynthetic genes responsible for synthesizing the IGS core structure, or CYP81F2, the enzyme catalyzing core structure modification to 4MI3G, significantly reduced mild osmotic stress- and MKK9 (DD) -induced 4MI3G. Thus, our study demonstrates that mild osmotic stress promotes 4MI3G biosynthesis and the accumulation in Arabidopsis through activation of the MKK9-MPK3/MPK6 cascade and provides an MAPK-mediated signaling pathway for the IGS response to abiotic stress in plants.