Mechanisms Of Nitrogen And Phosphorus Acquisition Coordinated By STOP1 In Arabidopsis

Phosphorus is an indispensable macronutrient required for plant growth and development.Although total soil phosphorus is generally high,most part of the phosphorus is immobile and not available for uptake by plant.Phosphate is rapidly immobilized owing to fixation and microbial activity when it is applied as fertilizer in soils,and natural phosphate reserves are finite.One of the common strategies for crops to adapt to phosphate deficiency is root system architecture remodeling,mainly including repressed primary root elongation,which was thought to help in forging phosphorus from topsoil rich in phosphate resources,but the upstream signal involved in phosphate starvation responses remains unknown.On the other hand,ammonium has long been known to enhance phosphate acquisition efficiency in agriculture;however,the molecular basis is unclear.In addition,the uptake of ammonium by plants is strictly controlled at the transcriptional and post-transcriptional levels,but currently there are very few upstream regulatory factors identified.Using the model plant Arabidopsis thaliana as the research material,we preliminarily explored the role of ammonium in phosphate deficiency-mediated root system architecture remodeling,and conducted a preliminary exploration of the molecular mechanism of its improved utilization of insoluble phosphate.Also,we conducted an in-depth analysis of the regulaory factors of ammonium absorption.Here,we discovered that a zinc finger transcription factor STOP1(Sensitive to proton rhizotoxicity 1)acts as a core regulator for the integration of nitrogen and phosphorus nutrition signals and uptake.1.Ammonium uptake regulated by ammonium transporters determines root system architecture remodeling under phosphate deficiencyWe found that the Arabidopsis root system remodeling caused by low phosphate requires the presence of ammonium.The main root elongation of the ammonium transporter quadruple knockout mutant qko amt1;2 amt1;3 amt2;1)is not inhibited under low phosphorus,and the number of lateral roots and root hair density are not significantly increased.Perls/DAB staining results showed that the repressed main root growth under low phosphate was caused by ammonium regulated excessive accumulation of Fe3+in the roots,and this Fe3+over-accumulation was significantly inhibited in the roots of qko mutant.Interestingly,the inhibition of main root elongation by ammonium under low phosphate was disappeared in stop1 mutant.The staining analysis found that the excessive accumulation of Fe3+ in roots enhanced by ammonium under low phosphate was STOP1 dependent.RT-qPCR analysis results showed that ammonium activated the expression of the malate transporter gene ALMT1,a downstream gene of STOP1,under phosphate deficiency,while the qko mutant could not induce expression of ALMT1.In summary,phosphate deficiency activates the STOP1 signaling pathway through ammonium uptake regulated by ammonium transporters,which affects the accumulation of Fe3+ in the root tip,thereby mediating changes in root system architecture.2.Rhizosphere acidification caused by ammonium uptake activates the STOP1 signaling pathway under low phosphate stressRhizosphere acidification is one of the common strategies for plants to cope with phosphate deficiency,which can improve the availability of insoluble phosphate in the soil.We found that the phenomenon of rhizosphere acidification promoted by low phosphate can only be observed in the media supplied with ammonium;additionally,rhizosphere acidification cannot be observed in qko mutants,indicating that low phosphate induced rhizosphere acidification is triggered by ammonium absorption.Moreover,we found that ammonium specifically activates the accumulation of STOP1 protein at the post-transcriptional level in response to phosphate deficiency,and the accumulation of STOP1 can also be accumulated when the medium pH is low enough.Interestingly,when the pH of the medium is buffered by MES,STOP 1 protein accumulation induced by ammonium was greatly reduced.Taken together,these results show that ammonium uptake regulated by ammonium transporters promotes rhizosphere acidification under low phosphate,which triggers the accumulation of STOP 1 protein,and finally the iron homeostasis affected by malate efflux leads to serious main root growth inhibition.3.Ammonium-STOP1-organic acid module improves the utilization of insoluble phosphateGiven that ammonium can activate the accumulation of STOP 1 protein under phosphate deficiency,we further analyzed the effects of ammonium and nitrate on the utilization of insoluble phosphate by plants.In order to avoid the inhibition of main root growth indirectly caused by light exposure,and also to better mimic the habit of roots growing in darkness under natural conditions,we analyze the utilization of insoluble phosphate with plant root under shading treatment.The results showed both the wild-type and stop1 mutants showed obvious phosphate starvation disease when nitrate as the sole nitrogen source,and growth between the two was basically the same;however,in the presence of ammonium,whether it was added Ca₁₀(PO₄)₆(OH)₂ On the medium of FePO₄,plant growth was significantly improved compared with the sole nitrate culture when supplied with Ca₁₀(PO₄)₆(OH)₂ or FePO₄ in the medium.What is interesting is the growth and phosphate content of the wild-type and stop1 mutants was nearly the same on the medium supplemented with Ca₁₀(PO₄)₆(OH)₂,but on the medium treated with FePO₄,the growth and phosphate content of the wild-type was significantly better than that of the stop1 mutant.Considering the similar rhizosphere acidification ability of the wild-type and stop1 mutant,ammonium-improved phosphate utilization on Ca₁₀(PO₄)₆(OH)₂ medium should be attributed to the dissolution caused by acidification,while the utilization of FePO₄ was partially dependent on the STOP1-ALMT1 pathway mediated by ammonium absorption.4.STOP1-CIPK23 signal inhibits ammonium absorptionExcessive application of ammonium fertilizer may adversely affect the growth of plants.We found that under phosphate sufficient condition,high ammonium treatment can also activate the expression of STOP1 protein.The STOP1 loss-of-function mutant exhibited hypersensitivity to ammonium stress.RT-qPCR analysis found that the expression of the ammonium transporter gene AMT1s in stop1 mutant was significantly up-regulated.The ¹⁵N-labeled ammonium uptake rate measurement showed that the stop1 mutant had a higher ammonium uptake rate.The expression of protein kinase CIPK23 is rapidly induced by excess ammonium,which could phosphorylate the ammonium transporters AMT1;1 and AMT1;2,thereby inhibiting ammonium absorption activity.Here,we found that the expression of CIPK23 in stop1 mutant was significantly reduced,and STOP1 can directly bind to the promoter region of CIPK23 and activate its expression.In summary,STOP1 triggers the expression of CIPK23,which in turn represses ammonium transporter activity,to avoid toxicity in response to excess ammonium.