Study On The Mechanism Of PHO85 Participating In Cellular Nutritional Metabolism Through TOR Pathway

Nitrogen is one of the basic elements of cells.It participates in the basic processes of protein,nucleic acid and energy metabolism,and is the main limiting factor of biological production.In recent years,a large amount of nitrogen fertilizer has been applied to improve crop yield,resulting in serious non-point source pollution.It is an effective way to solve the abuse of nitrogen fertilizerto study the mechanism of cell transformation and utilization of nitrogen,and cultivate nitrogen efficient crops.In this paper,based on our previous discovery that PHO85 can tolerate low nitrogen environment,its low nitrogen tolerance mechanism was further studied.Firstly,pho85? was found to have a better growth advantage than BY4741 in low nitrogen environment.Further analysis showed that pho85? tolerance to low nitrogen was achieved by escaping the restriction of leucine concentration in cells.WHI2 is a key gene that can respond to leucine,and whi2? is also tolerant to low nitrogen environment.Genetic complement analysis indicated that PHO85 was located upstream of WHI2 and could regulate the expression of WHI2.Pho85 p is a cyclin dependent kinase with ten cyclins downstream.Genetic analysis showed that PCL2 and CLG1 genes play a key regulatory role in PHO85 mediated low nitrogen tolerance.Leucine is a mediator of TOR sensing the adequacy of cellular nitrogen source,and SFP1 p is the main effector of TORC.When leucine is abundant,it activates TOR,which promotes phosphorylated SFP1 to enter the nucleus and activates the transcription of ribosomal protein genes.When leucine is deficient,SFP1 is dephosphorylated and exits the nucleus,inhibiting the transcription of ribosomal protein genes.We found that,even in low nitrogen environments,SFP1 was localized in the nucleus and up-regulated the expression of RPS3,a ribosomal protein gene activated by phosphorylated SFP1.The results showed that PHO85 was a negative regulator of TOR,and it could regulate the expression of WHI2 and inhibit the activity of TOR in low nitrogen environment.In addition,fluorescence observation showed that pho85? and whi2? exhibited stronger autophagy than wild-type strains,suggesting that pho85? and whi2? promoted autophagy to increase nitrogen use efficiency and tolerance to low nitrogen environment.Secondly,leucine,as one of the branched-chain amino acids,is a conditional essential amino acid for yeast.We tried to explore the regulatory mechanism of pho85? and whi2? activatiing TOR by branched-chain amino acid metabolism pathway.The removal of the other two branched chain amino acids(valine,isoleucine)or glutamate in low nitrogen medium could eliminate the growth advantage of pho85? and whi2? over the wild strains.We hypothesized that the growth advantage of pho85? and whi2? in low nitrogen environment was related to the branched-chain amino acid transamination reaction(BAT1)participated by Glu.By RT-PCR,we found that the expression level of BAT1 was significantly up-regulated in pho85? and whi2? strains in low nitrogen environment compared with wild strains.In the TCA cycle,the interaction of BAT1 p and ACO1 p promote the flux of the TCA cycle.In order to verify whether the activation of pho85?and whi2? is related to the TCA cycle,we constructed two mutants of pho85?aco1? and whi2?aco1?.They no longer had better growth advantages in low nitrogen environment.Meanwhile,the destruction of mitochondrial function of pho85? could also eliminate the growth advantage of pho85? in low nitrogen environment,suggesting that the low nitrogen tolerance of pho85? was dependent on the mitochondrial and TCA cycles.Then the mitochondrial membrane potential and ATP levels of pho85? and whi2? were measured.The data showed that pho85? and whi2? activated TOR pathway by promoting TCA cycle and increasing ATP content.The growth advantage of pho85? and whi2? could be inhibited by removing glucose or adding2-deoxyglucose in low nitrogen media.When glucose was replaced by glycerol or ethanol,the growth of wild strains in low nitrogen could be restored,further confirming that PHO85 and WHI2 participate in the negative regulation of TOR pathway by inhibiting the initial stage of glycolysis.In summary,we found that PHO85 was a negative regulator of TOR,located upstream of the WHI2 gene,which was regulated by the concentration of leucine in low nitrogen environment,thus inhibiting TOR.Meanwhile,PHO85 could inhibit the flux of TCA cycle by inhibiting the BAT1 of leucine transaminase and the initial stage of glycolysis in low nitrogen environment,leading to the inhibition of TOR.The results revealed the mechanisms of Pho85-mediated low-nitrogen tolerance and provided basic data for the cultivation of nitrogen efficient crops.