Identication Of A Novel Aluminum Resistance Gene ArPk And Involved Mechanisms

Aluminum toxicity is one of the main limiting factors for rice yield in acid soil.As the most Al resistance cereal crop,rice Al resistance mechanisms around the key regulation factor ART1 have been comprehensively elaborated.However,whether there are other regulation mechanisms independent of the ART1-regulated Al resistance mechanism remains to be explored.Nitrogen is one of the necessary macronutrients for plant growth.Long-term excessive application of nitrogen fertilizers such as urea,ammonium carbonate or ammonium bicarbonate can cause soil acidification,thus increasing Al activity in the soil and arousing Al toxicity in plants.Nevertheless,it is still unclear whether nitrogen affect plant Al resistance through nitrogen metabolites.In order to discover new Al resistance genes,we screened a rice EMS mutant library in the Oryza sativa indica cv.Teqing background and isolated an Al sensitive mutant.Through BSA based Mut Map,physiological analysis,transcriptome analysis et al.,we confirmed the mutation in a protein kinase gene caused rice sensitive to Al,thus directly affecting polyamine biosynthesis,which is part of nitrogen metabolism and have a close relation with Al sensitivity.Detail research results are as follows:1.A mutation in the plasma membrane and nucleus dual-localized proteinkinase Ar PK caused Al sensitive phenotype in the mutant.An Al-sensitive mutant obtained from screening the rice EMS library that exhibits significant growth inhibition under 5?M Al treatment,but its response to heavy metals such as Cd²⁺,Zn²⁺,Cu²⁺,La³⁺and different p H treatments were not significantly different from that of the wild type.Through Mut Map we got several candidate genes and the Al-sensitive phenotype of the mutant was reverted after transferred a candidate kinase gene,indicating the kinase gene is involved in rice Al resistance regulation;therefore,we named the gene Ar PK(Aluminum related Protein Kinase).Through GUS staining and gene expression detection,it was found that Ar PK was significantly induced under short-term high Al concentration(50?M,3 h)or long-term low Al concentration treatment(20?M,24 h).Through in situ immunofluorescence staining and examining tobacco transient expression,we found that Ar PK was localized in the plasma membrane and nucleus.By examining expression of the key transcription factor ART1 that regulated rice Aluminum resistance and its downstream Al resistance genes,we found that the expression of these genes in arpk was not significantly different from that of the wild type,which suggested that Ar PK was involved in a novel Aluminum resistance signaling network independent of ART1-regulated Al resistance mechanism.2.Rice NAOGAc T may compensate for the function of NAODTranscriptome analysis revealed that the expression of a gene encoding acetylornithine deacetylase(NAOD)was constitutively suppressed in the mutant.However,transferring Ca MV35S promoted NAOD into arpk could not relief the Al toxicity phenotype,indicating that NAOD transcriptional repression is irresponsible for the Al-sensitive phenotype in arpk.NAOD is a dependent enzyme for the linear ornithine synthesis pathway,catalyzing conversion of N-acetyl-Orn to Orn.Considering that in some prokaryotes and higher plant there is a cyclic pathway for ornithine synthesis,we found that NAOGAc T,which transfer acetyl residue of acetylornithine to glutamate through transacetylation thus generate Orn and another product acetylglutamate enters the circulation in cyclic ornithine synthesis pathway,was expressed normally and up-regulated by Al in arpk.In addition,high ammonium addition alleviated the Al toxicity in arpk,and high ammonium plus Al treatment resulted in higher NAOGAc T expression of in arpk than single Al treatment.These results suggested that both linear pathway and cyclic pathway for ornithine synthesis exist in rice,and NAOGAc T can compensate for the function of NAOD under certain conditions.3.Put synthesis disorder in arginine metabolism affects arpk Al resistanceTranscriptome analysis revealed that the encoding gene of the enzyme ODC,which is required for the next catalytic step after NAOD catalyzed Orn generation,is specifically,significantly up regulated by Al in arpk,inferring an inhibitor role of Ar PK.ODC catalyzes the synthesis of putrescine(Put)from Orn,and Put is a molecule with multiple functions and is reported to be involved in a variety of plant stresses.Detection of Put content showed that the content of the three forms of Put(free,bound and bound)in arpk were all induced by Al,and was significantly higher than the that in WT.After apply of the ODC inhibitor DFMO,the transcriptional inhibition of NAOD in arpk was relieved and the Al-sensitive phenotype of arpk was restored.The results suggested that the transcriptional inhibition of NAOD in arpk may be a negative feedback on the release of ODC1 inhibition caused by the mutation in Ar PK and accumulation of Put caused by the excessive up-regulation of ODC1 is the reason for arpk Al-sensitive phenotype.The biosynthesis of Put in rice has two pathways:ODC pathway and ADC pathway.However,despite up regulated expression of ADC2 in WT to Al,ADC inhibitor DFMA had little inhibition effect on WT root elongation whereas ADC inhibitor D-Arg showed no repression.Given this,we believe that ADC2 may play a limited role in rice Al tolerance,while stably regulating of ODC dependent polyamine synthesis is the basic strategy for rice Al tolerance.4.Ammonium improves the arpk Al resistance by increasing its assimilation and inhibiting ODC transcription thus reducing Put synthesisWe further analyzed the reason for the Al detoxification in arpk.We found that Al+10m M NH₄⁺treatment increased the expression of most genes in the GS/GOGAT pathway and arginine synthesis pathway,while inhibited ODC1,ADC2 and SAMDC1 expression in polyamine synthesis.This implied that the high concentration of NH₄⁺in arpk on the one hand inhibited the transcription of ODC and reduced the synthesis and accumulation of Put;and on the other hand,it may increase intracellular amino acids(Glu,Gln,etc.)and other Al resistance proteins by enhancing NH₄⁺assimilation;the joint effect of the two aspects resulted in the alleviated Al toxicity of arpk.