Functional Study Of GmSTOP1s Gene Family In Soybean Adaptation To Low Phosphorus,Acid And Aluminum Stress

More than 40%of the world cultivable soil is acidic.Low phosphorous(P),acid stress and aluminum(Al)toxicity are primary constraints to crops production on acid soils.In order to adapt to these stresses,plants have developed a series of morphological,physiological and molecular mechanisms.In Arabidopsis,the key node gene,At STOP1,plays a crucial role in adaptation to low phosphorus,acid stress and Al toxicity.However,the function of STOP1 in legumes in regulating root system adaptation to these stresses is not clear.Therefore,the present study choose soybean as the research object.Three GmSTOP1s homologous were identified in the soybean genome.The functional similarities and differences of these three GmSTOP1s homologous in adaptation to acid stress and Al toxicity were systematically compared.Subsequently,functional analysis of GmSTOP1-3 was conducted using both Arabidopsis Atstop1 mutant with GmSTOP1-3 complementation and transgenic soybean plants with GmSTOP1-3 overexpression.The root morphological changes and the regulation network of phosphorus signals were investigated in these transgenic materials.Furthermore,the candidate proteins interacting with GmSTOP1-3 were obtained through yeast dual library screening.The main results are shown as follows:1,Comparative analysis of the function of GmSTOP1s family genes in soybean adaptation to acid stress.Expression patterns analysis showed that the expression of three GmSTOP1s were not affected by acid stress.Furthermore,transgenic Arabidopsis Atstop1mutant with GmSTOP1s over-expression were used for functional analysis of GmSTOP1s in response to acid stress.The results showed that all three genes,GmSTOP1-1,GmSTOP1-2and GmSTOP1-3,were able to reverse the H~+sensitivity of the Atstop1 mutant.In addition,the expression of H~+tolerance genes,including GDH1,GDH2,GABA-T and At NADP-ME2,which play a role in maintaining the p H homeostatic state of plants,were partially restored in complemented lines of three genes under acid treatment.2,Comparative analysis of GmSTOP1s family genes in soybean adaptation to Al toxicity:Expression pattern analysis showed that the expression of both GmSTOP1-1 and GmSTOP1-3 in root apexes was up-regulated by Al toxicity,which increased with the increase of Al concentration.However,the expression of GmSTOP1-2 was not affected by Al treatments.Furthermore,complementation assays demonstrated that GmSTOP1-1 and GmSTOP1-3could partially recover the tolerance of root growth of Atstop1 mutants in response to Al toxicity.In addition,under Al treatment,complementary expression GmSTOP1-1 or GmSTOP1-3 could partially restore the expression of Al-resistant genes,such as PMI,At TDT,NADP-ME2 and At MATE.3,Functional analysis of GmSTOP1-3 in soybean responsive to P deficiency.Functions of GmSTOP1-3 were further examined in soybean transgenic lines with GmSTOP1-3overexpression.It was found that GmSTOP1-3 overexpression significantly increased lateral root length of transgenic soybean plants.Subsequently,RNA-seq was used to analyze changes of gene expression profiles of soybean roots between wild type and the transgenic lines under both high P and low P conditions.The results showed that overexpressing GmSTOP1-3 led to increased transcripts of 108 genes in soybean roots,in which a set of genes were involved in the organic acid secretion and nutrient transportation.The results suggested that GmSTOP1-3 might affect soybean root growth and development by regulating organic acid secretion and nutrient transportation.4,Screening and verification of GmSTOP1-3 interacting proteins.A total of seventeen proteins were found to be interacted with GmSTOP1-3 by yeast two-hybrid.The interaction between GmSTOP1-3 and CAX-interacting protein was further verified by yeast two-hybrid and bimolecular fluorescence complementation(Bi FC).In summary,this study systematically analyzed the biological functions of soybean GmSTOP1 members in root responses to acid stress and Al toxicity.The results showed that all three GmSTOP1s were functionally conserved in response to acid stress,while have divergent functions in response to Al toxicity.Subsequently,We analyzed the functions analysis of GmSTOP1-3 was conducted using transgenic soybean plants.Results showed that overexpression of GmSTOP1-3 increased the lateral root length of transgenic plants.Transcriptome analysis revealed that overexpression of GmSTOP1-3 changed the transcription levels of a serial genes such as organic acid transporter and nutrient transporters,which might be involved in soybean roots adaptation to P deficiency.The results of this study laid a theoretical foundation for cultivating soybean varieties that were synergistically adapted to low phosphorus,acid stress and Al toxicity in acidic soil.