Study On Maize Cytoplasmic PPDK Promoting The Activity Of Starch Synthesis Rate-limiting Enzyme AGPase

Maize(Zea Mays L.)is one of the three major crops in the world,and it is an important dual-purpose crop in production and life.Starch is an important industrial raw material with extensive production and utilization,involving more than 30 fields,covering all walks of life.Starch is the most widely used substance in corn,accounting for about 70% of the dry weight of corn kernels,and is an important determinant of corn yield and quality.Generally,starch is synthesized in the seed endosperm of cereal crops.The starch biosynthesis process is a complex process regulated by a series of key enzymes of starch synthesis,and the current research is not perfect.Among these key enzymes,AGPase,which catalyze the formation of the ADPG,is often described as the rate-limiting enzyme of the starch biosynthesis pathway in cereals,as it has significant control over the glucose precursor require for starch synthesis.Cytosolic and plastidial AGPase enzymes exist in cereal endosperm,but 85-95% of the AGPase activity in corn endosperm is mainly derived from the cytosolic AGPase.AGPase activity determines endosperm starch level in cereals,and consequently the yield and quality of cereal kernels.In cereal,AGPase activity is regulated by various mechanisms including transcriptional regulation,protein modification regulation,regulation by sugar and ABA signaling,among others.Moreover,some studies have reported regulation of key enzymes via multi-enzyme complexes or interaction of starch metabolic enzymes with non-enzyme proteins.However,whether AGPase participates in multi-enzyme complex with other metabolic key enzymes or non-enzyme proteins has not yet been reported.This study aimed to explore the interaction between AGPase and other proteins in maize endosperm,study the effect of protein interaction of cytosolic AGPase on its activity in maize endosperm and its application on maize production.The specific research results are as follows:1.There was interaction between cytosolic AGPase and PPDK in maize endosperm.Using the endosperm of the maize inbred line W64 15 days after pollination as the material,the total endosperm protein was extracted,and the endosperm cytoplasmic AGPase large and small subunit SH2 and BT2 specific antibodies were used for co-immunoprecipitation,and the co-precipitated protein was detected by mass spectrometry.It was shown that pyruvate phosphate dikinase(PPDK)was identified in the protein samples precipitated by SH2 or BT2 antibody,indicating that PPDK may interact with the cytosolic AGPase in maize endosperm.The total protein of endosperm was co-immunoprecipitated with PPDK-specific antibody and detected by mass spectrometry,and SH2 and BT2 were also detected,which further confirmed the interaction between PPDK and cytosolic AGPase in maize endosperm.Using BT2 and PPDK antibodies to conduct one-to-one verification by Co-IP technology,the results showed that there were obvious bands in the input and IP results of the two groups of experiments,which further confirmed the interaction between PPDK and cytosolic AGPase.2.Prokaryotic expression of cytoplasmic PPDK and its enzymatic properties.The maize cytosolic PPDK prokaryotic expression vector was constructed,and the enzyme activity was detected by prokaryotic expression,purification and detection.The results showed that the prokaryotic expression protein of maize endosperm cytosolic PPDK was highly expressed at 28°C after induction for 4-6h,the protein molecular weight was about126 k Da,and the protein was mainly soluble protein.It shows that the prokaryotic expression of maize endosperm cytosolic PPDK has enzymatic properties and can be used for in vitro molecular experiments.3.The dynamics of SH2,BT2,PPDK accumulation and PPDK activity of maize inbred line W64 during grain filling.The accumulation of PPDK,SH2 and BT2 in different filling stages of maize inbred line W64 was detected by Western Blot.The results showed that PPDK exhibited low accumulation level during the early stage of endosperm development,and but was present at higher concentration during the mid and late stages of endosperm development.Similarly,SH2 and BT2 also showed low and higher levels of accumulation during the early and mid to late stages of endosperm development.PPDK activity was low during the early and late stages of grain filling,but was higher during the mid-stage of grain filling,and peaked at 18 DAP.4.PPDK affects the activity of cytosolic AGPase in maize endosperm.The endosperm crude enzyme solution was extracted from W64 9 days,12 days and 18 days after pollination.Additionally,PPDK protein was expressed by exogenous addition of prokaryotic cells,and AGPase activity was detected after co-incubation.The results showed that exogenous addition of PPDK significantly increase the activity of cytosolic AGPase in the crude enzyme solution at 12 days after pollination compared to the control group,while the crude enzyme solution at 9 and 18 days after pollination had no significant change in the activity of cytosolic AGPase compared with the control group.This indicated that the accumulation and activity of PPDK in the endosperm of maize could affect AGPase activity in the maize endosperm,thereby affecting the rate of starch synthesis.In conclusion,this study found the interaction between cytosolic AGPase and PPDK in maize endosperm.PPDK has a positive effect on activity of cytosolic AGPase in maize endosperm.The study provides new information on the regulatory networks of AGPase activity in maize,and also provide theoretical basis for maize improvement.It provides a new idea to improve the AGPase activity of crops,increase the rate of starch synthesis,and generate economic and social benefits in production.