Molecular Mechanism Of MYB4-MAN3-Mannose-MNB1 Signaling Cascade In Regulation Of Cadmium Accumulation And Tolerance In Arabidopsis

Our previous studies showed that MAN3-mediated mannose plays an important role in plant responses to cadmium(Cd)stress.However,the underlying mechanisms and signaling pathways involved are poorly understood.In this study,we showed that an Arabidopsis MYB4-MAN3-Man-MNB1 signaling cascade is involved in the regulation of Cd accumulation and tolerance.Loss-of-function of MNB1 led to decreased Cd accumulation and tolerance,whereas overexpression of MNB1 significantly enhanced Cd accumulation and tolerance.Consistently,expression of the genes involved in the GSH-dependent phytochelatin(PC)synthesis pathway(such as GSH1,GSH2,PCS1,and PCS2)was significantly reduced in the mnb1 mutants but markedly increased in the MNB1-OE lines in the absence or presence of Cd stress,which was positively correlated with Cd-activated PC synthesis.In addition,we found that mannose can specifically bind to MNB1 and this binding is dependent on MAN3 mediated cadmium tolerance.Further analysis showed that MYB4 can directly activate the transcription of MAN3,and MYB4 gene also positively regulates the accumulation and tolerance of cadmium by GSH-dependent PC synthesis pathway.A series of double-mutant materials were further constructed.Through phenotypic analysis,it was found that overexpression of MAN3 restored the cadmium-sensitive phenotype of the myb4 mutant but failed to restore the cadmium-sensitive phenotype of the mnb1 mutant,whereas overexpression of MNB1 can restore the cadmium sensitive phenotype of the myb4 mutant.Together,these results indicate that MAN3 acts downstream of MYB4 but acts upstream of MNB1.Taken together,our results provide compelling evidence that a MYB4-MAN3-Man-MNB1 signaling cascade regulates cadmium accumulation and tolerance in Arabidopsis through the GSH-dependent PC synthesis pathway.To some extent,this study reveals the mechanism of plant regulation by multiple genes on heavy metal stress and our study results provide more theoretical basis for plant restoration.