Functional Analysis Of Protein Kinase ZmCyPPDK1 In Response To Drought Stress In Maize Seedlings

Drought stress seriously affects plant growth,and even causes plant death.Maize is an important food crop,however,the growth and yield of maize are seriously affected by drought stress.Drought stress could cause oxidative damage,oxidative damage consists of reactive oxygen species（ROS）,and H₂O₂is an important member of ROS.H₂O₂could destroy cell membranes,damage organelles,damage DNA,and eventually cause the collapse of the cell system,leading to the death of the plant.In order to cope with oxidative damage,plants have evolved antioxidant systems,including enzymatic system and non-enzymatic system.The enzymatic system plays an important role in plant resistance to oxidative damage.The enzymatic system mainly consists of catalase（CAT）,ascorbate peroxidase（APX）and superoxide dismutase（SOD）.Among them,CAT and APX are the key enzymes to remove H₂O₂,and improving the activity of CAT or APX could remove H₂O₂more effectively.Protein kinases could improve CAT activity by phosphorylating CATs,and then accelerate the decomposition of H₂O₂,and finally improve the tolerance of plant.Pyruvate orthophosphate dikinases（PPDKs）belong to protein kinase,and PPDKs involved in a wide range of plant growth and development processes.However,the mechanism through which PPDK participates in these processes remain unclear.In maize,Zm PPDKs are encoded by ZmCyPPDK1,ZmCyPPDK2 and Zm C₄PPDK1.In this study,maize inbred lines B73 were used as background materials to construct maize genetic materials with overexpression and RNAi interference of Zm PPDKs,and the T₂generation materials of Zm PPDKs were treated with drought stress,the results showed that ZmCyPPDK1 could improve maize tolerance to drought stress by reducing H₂O₂content.We conducted a further study on how ZmCyPPDK1 regulates H₂O₂content,and the specific results are as follows:1.ZmCyPPDK1 and Zm C₄PPDK1 could improve maize tolerance to drought stress by reducing oxidative damage.However,ZmCyPPDK2 could not improve the tolerance in maize to drought stress.ZmCyPPDK1 could remove excessive H₂O₂by regulating the activity of CAT,Zm C₄PPDK1 could not remove excessive H₂O₂by regulating the activity of CAT.2.In maize,Zm CATs are encoded by Zm CAT1,Zm CAT2 and Zm CAT3,ZmCyPPDK1could interact with Zm CAT1,Zm CAT2 or Zm CAT3 in yeast,and ZmCyPPDK1 could improve the activity of Zm CAT2.ZmCyPPDK2 and Zm C₄PPDK1 could not interact with Zm CAT1,Zm CAT2 or Zm CAT3 in yeast and could not enhance their activity.3.ZmCyPPDK1 phosphorylated Zm CAT2 at Thr-293 and enhanced its activity.Drought stress induced up-regulation of ZmCyPPDK1 kinase activity.Under drought stress,Thr phosphorylation and activity of Zm CAT2 increased significantly.Further studies showed that drought stress enhanced the thr phosphorylation and activity of Zm CAT2 by ZmCyPPDK1.4.To investigate whether Zm CAT2 could improve tolerance to drought stress in maize,maize inbred lines B73 were used as background materials to construct maize genetic materials with overexpression and RNAi interference of Zm CAT2,and the T₂generation materials of Zm CAT2 were treated with drought stress.The results showed that Zm CAT2improved maize tolerance to drought stress by reducing oxidative damage.To further explore whether Thr-293 site could enhance Zm CAT2 tolerance to drought stress,maize inbred lines B73 were used as background materials to construct maize genetic materials with overexpression of Zm CAT2^T293Aand Zm CAT2^T293D,and the T₂generation materials of Zm CAT2^T293Aand Zm CAT2^T293Dwere treated with drought stress.The results showed that,after drought treatment,when compared with OE-Zm CAT2 plants,OE-Zm CAT2^T293Aplants had poor growth,OE-Zm CAT2^T293Dplants had better growth,suggesting that Thr-293enhanced Zm CAT2 tolerance to drought stress.5.To further study the function of ZmCyPPDK1,and its interaction protein Zm MYB-CC10 was obtained,and found that ZmCyPPDK1 could interact with Zm MYB-CC10.Then,to study whether Zm MYB-CC10 could improve drought tolerance in maize,maize inbred lines B73 were used as background materials to construct maize genetic materials with overexpression of Zm MYB-CC10,and the T₂generation materials of Zm MYB-CC10 were treated with drought stress.The results showed that Zm MYB-CC10directly regulated the expression of Zm APX4,increased the activity of APX,reduced the accumulation of H₂O₂,alleviated oxidative damage,and ultimately improved the drought tolerance in maize.In summary,ZmCyPPDK1 regulates the activity of Zm CAT2 by phosphorylating Zm CAT2 at Thr-293,thereby improving the activity of CAT,reducing the content of H₂O₂,alleviating oxidative damage,and ultimately improving the drought tolerance in maize.ZmCyPPDK1 could interact with Zm MYB-CC10,Zm MYB-CC10 directly regulates the expression of Zm APX4,enhances the activity of APX,reduces the accumulation of H₂O₂,and enhances the tolerance in maize to drought stress.