Investigation On Molecular Mechanism Of ZmPHT1;9 Induced By Arbuscular Mycorrhizal Fungi To Promote Absorption Of Phosphate In Maize

Phosphorus is an indispensable class of major mineral elements in plant growth and metabolism.It is an essential component of phospholipids,nucleic acids,ATP and so on.However,the content of inorganic phosphorus in soil that can be planted is very low.In order to cope with low phosphate environment,plants evolved some strategies,and forming symbiosis between plant and arbuscular mycorrhizal(AM)fungi is one of these strategies.At present,AM fungi play an important role in promoting plant uptake of nutrient elements,and their research has been paid more and more attention.Maize(Zea mays L.)is an important crop,and low phosphorus stress seriously affects its yield.The current research status shows that AM fungi can effectively promote the absorption of phosphorus in maize,but lack of in-depth study of the relevant regulatory mechanisms.PHT1 transporters(Phosphate Transporter 1)could improve the phosphate uptake efficiency(PAE)and phosphate utilization efficiency(PUE).Therefore,find PHTls in maize and study the molecular regulation mechanism being important for crop breeding technology and the sustainable development of modern agriculture.In this study,it has been found that PHT1 plays an important role in symbiosis uptake and transport of phosphate in plants.We focused on the metabolism of phosphate in the process of phosphate starvation and AM fungi symbiosis in maize.The ZmPHT1 genes of maize B73 genome were identified by bioinformatics.And the functions of ZmPHT1;9 in phosphate absorbtion and transporte were studied by overexpressing in plants and mutant yeast complementry experiment.Further molecular mechanism was studied by yeast one-hybrid,yeast two-hybrid and bimolecular fluorescence complementation.The main results of the study are as follows:1.Maize B73 and AM fungi were used as experimental materials to study the phenotype,phosphorus content of symbiotic maize.Datas showed under low phosphate stress,inoculation with AM fungi could significantly promote the growth of maize roots,increase the content of phosphorus in both roots and leaves.2.A total of 13 non-repeating PHT1 phosphate transporter genes were identified on the whole genome level of maize B73,named as ZmPHT1;1-ZmPHT1;13.Under low phosphate stress,ZmPHT1;2,ZmPHT1;4,ZmPHT1;6,ZmPHT1;7,ZmPHT1;9 and ZmPHT1;11 were induced in roots with AM fungi inoculation.Tissue expression pattern analysis revealed that only ZmPHT1;1 ZmPHT1;9 and ZmPHT1;13 were expressed in roots under normal conditions.In the complementary experiment of yeast phosphate deficient mutant,we found that 13 ZmPHT1 genes can completely or partially complement the phenotype of the yeast mutant.The results indicated that 13 PHT1 genes of maize had the function of promoting phosphate uptake.3.Subcellular localization of ZmPHT1;9 showed it was located in the cell membrane of plant.The functions of candidate gene ZmPHT1;9 were studied by complementary experiment of yeast phosphate deficient mutants and overexpression transgenic rice.ZmPHT1;9 can effectively complement the loss phosphate transport function of yeast phosphate deficient mutants,improve the absorption of phosphate and promote growth of yeast mutant under low phosphorus conditions.In transgenic rice,under low phosphate environment,overexpression plants have more growth advantages than wild type,as well as significantly higher amount of phosphate uptake than that of wild type.In addition,genes related to low phosphate response in transgenic plants showed a decreasing trend.Combined with the above studies,ZmPHT1;9 has the functions of promoting phosphate uptake and affecting plant growth.4.The effects of AM fungi on ZmPHT1;9 were analyzed by constructing the symbiosis system of AM fungi and transgenic Lotos with pZmPHT1;9-GFP and pZmPHT1;9::ZmPHT1;9-GFP.The results suggested that ZmPHT1;9 was induced by AM fungi and it was localized on the surrounding membrane of the arbuscular,which was presumed that ZmPHT1;9 had a role in the surrounding membrane.5.The molecular mechanism of AM fungi regulating ZmPHT1;9 was further analyzed.The relationship of candidate transcription factors were analyzed by yeast one-hybrid,yeast two-hybrid and bimolecular fluorescence complementation experiments.Through the above analysis,we speculate that under low phosphate stress,AM fungi controled ZmHAM2,ZmD9,ZmSHR1 and ZmHAM1 forming a four-protein complex combined with ZmPHR-like,activating the expression of ZmPHT1;9,which can promote the absorption of phosphate in maize.