Preliminary Study On The Function Of GmNTLs In Soybean Adaptation To Phosphorus Deficiency And Aluminum Toxicity

More than 40% of the world’s potentially arable lands are composed of acid soils,and the area exceeds 20 million hectares in China.Phosphorus(P)deficiency and aluminum(Al)toxicity have become major factors threating crop production on acid soils.NTL proteins are a group of NAC transcription factors,and play an important role in the mechanisms of plant response to various abiotic stresses,such as drought,salt and cold stress.However,the underlying adaption mechanism of whether NTL is involved in regulating low P deficiency and Al toxicity in plants remain poorly understood.Soybean is important grain and oil crop.Therefore,this study focused onanalyzing the function of GmNTLs in soybean adaptation to P deficiency and Al toxicity.Bioinformatics analysis and expression pattern analysis were performed on 15 members of the GmNTL family in the soybean.At the same time,we preliminary analyzed the function of some members in the adaptation mechanism of low phosphorus and aluminum toxicity by overexpressing the genes in Arabidopsis thaliana.The main results as follows:1.Through homology comparison,we identified 15 GmNTL homologous genes in soybean genome.The phylogenetic analysis revealed that 15 GmNTL are divided into three subgroups.Structural analysis showed that the N-terminus containing a NAC DNA-binding domain of all GmNTLs,which is highly conserved.Whereas the Cterminal region with α-helical transmembrane domain is quite diverse.The analysis of4 GmNTL showed in that GmNTL2 was located in both nuclei,and GmNTL4,GmNTL7 and GmNTL10 were located at cell nuclei and plasma membranes.2.The expression of seven GmNTL genes including GmNTL1,GmNTL2,GmNTL3,GmNTL4,GmNTL5,GmNTL8 and GmNTL12 are significantly upregulated by P deficiency after 6 days of low P treatment.After 12 days of low-phosphorus treatment,the expression level of GmNTL genes except GmNTL6 and GmNTL14 were significantly increased.3.After 24 hours of aluminum treatment,the expression level of most GmNTL genes,except GmNTL9 and GmNTL13,was significantly increased in the soybean root tip(< 2 cm)by Al treatment.4.9 GmNTL genes were overexpressed in Arabidopsis.Results showed that under normal condition,the fresh weight of transgenic Arabidopsis overexpressed GmNTL4,GmNTL10 or GmNTL12 were significantly increased plants compared to control lines.Overexpression of GmNTL2 and GmNTL4 significantly increased the plant fresh weight under P deficiency;whereas,compared with WT,the plant fresh weight of transgene lines overexpressed GmNTL1 or GmNTL12 were significantly decreased.However,overexpression of the other 4 GmNTL homologous genes had no significant effect on the fresh weight in Arabidopsis under both normal and low P conditions.5.To test the root growth of transgenic Arabidopsis overexpressed 9 GmNTL genes respectively in response to phosphorus deficiency.The results showed that overexpression of GmNTL4 significantly increased the primary root length of transgenic plants under high phosphorus conditions.Under low phosphorus conditions,overexpression of GmNTL2 increased,whereas,overexpression of GmNTL12 decreased the primary root length of transgenic plants.However,overexpression of the other 6 GmNTL homologous genes had no significant effect on the growth of transgenic Arabidopsis under all phosphorus conditions.6.The aluminum tolerance of transgenic Arabidopsis with overexpression of 9GmNTL genes were further investigated.The results showed that after 48 hours of Al treatment,the absolute growth of roots of transgenic Arabidopsis plants overexpressing GmNTL1,GmNTL4 and GmNTL10 was significantly increased compared to wild type.However,overexpression of other GmNTL genes had no significant effect on the growth of transgenic Arabidopsis.7.Furthermore,we analyze the effect of overexpressing GmNTL4 on the expression levels of aluminum-tolerance related genes in transgenic Arabidopsis plants.The results showed that over-expression of GmNTL4 significantly reduced the expression levels of STOP1,TDT and NADP-ME2 genes in transgenic plants after 48 hours of Al treatment,but had no significant effects on the expression levels of ALMT1,ALS3 and STOP2.Taken together,this study investigated that the regulation of spatiotemporal expression patterns of GmNTL family by low phosphorus stress and Al toxicity,and explored the function of GmNTL family under these stresses.These results can provide theoretical base for further analysis about the biological mechanisms of soybean adaptation to P deficiencyand Al.