ZmWRKY4Is Involved In The Antioxidant Defense Modulated By ZmMPK5in Maize

When plants suffer from stress, the content level of H2O2is increased, which mediate further signal transduction. And the accumulation of ROS can activate MAPK. WRKY plays important role in the process of biotic and abiotic stress as a kind of transcription factor, but the research of WRKY in antioxidant defense is very less.The experiments mainly study the function of ZmWRKY4in antioxidant defense and the interaction between ZmWRKY4and ZmMPK5in maize. Gene expression analysis, enzyme activity assay, maize protoplast transient expression system together with yeast two hybrid and BiFC were used in the experiments.Maize plants are treated with different concentration of Cd. Antioxidant enzymes activity is determined and the gene expression of antioxidant enzymes together with ZmWRKY4is assayed. The result is that, antioxidant enzymes activity reach the maximam at the time of8h, and each gene expression are up-regulated.The CDS(coding sequence) area of ZmWRKY4gene was amplified by RT-PCR from maize leaves. To determine potential subcellular locations of ZmWRKY4, we inserted ZmWRKY4into the pXZP008vector, then transformed the construct (pXZP008-Zm WRKY4) into maize protoplasts. Images of YFP-ZmWRKY4fusion were captured with the confocal laser scanning microscope, which showed that ZmWRKY4were located to nuclear. Additionally, we transformed pXZP00S-Zm WRKY4into maize protoplasts to overexpress ZmWRKY4. The result is that, the gene expression and total activities of antioxidant enzymes SOD, APX are significantly increased. These suggest that ZmWRKY4may be involved in the antioxidant defense.ZmWRKY4was silenced using the RNA interfere technology, total activities and gene expression of antioxidant enzymes SOD and APX were reduced about half. After recovering treated, activities of the enzymes both recover a little, and genes expression recover to the control level, cAPX even higher than control. These results further suggst that ZmWRKY4was involved in the antioxidant defense. It has been studyed that ZmMPK5can be activated by Cd, and gene expression of transcription factor ZmWRKY4also can be increased by Cd. As a type of transcription factor, ZmWRKY4probably has interaction with ZmMPK5, finally, we confirm the interaction between them through yeast two-hybrid and BiFC.