Functional Analysis Of GmNla1/2/4 In Mediating Phosphorus Homoestasis In Soybean

Nitrogen limitation adaptation(NLA),a RING type E3 ubiquitin ligase,has been demonstrated to be involved in regulating nitrogen and phosphorus balance in Oryza sativa and Arabidopsis thaliana.But their functions in soybean(Glycine max)remain unkonw.In the present study,four members of GmNLA were identified and characterized in soybean genome,which was named after GmNLA1,GmNLA2,GmNLA3 and GmNLA4,respectively.Subsequently,their expression patterns in response to nitrogen(N)and phosphorus(P)deficiencies were analyzed by quantitative real-time PCR.Finally,functions of GmNLA1,GmNLA2 and GmNLA4 were characterized to be involved in regulating nitrogen and phosphorus balance in soybean.The main results are shown as follows:1.The interaction between N and P significantly affected both N and P concentration in soybean.After 14 d of low N treament,compared to those at sufficient N levels,concentrations of souble Pi and total P were significantly increased in soybean young leaves and roots under Pi sufficient condition,while concentrations of souble and total P were no difference under Pi deficiency conditions.Constrastingly,compared to those under Pi sufficient conditions,Pi deficiency significantly decreased total N concentration in soybean young leaves and roots under N sufficient conditions,while increased total N concentration in soybean young leaves and roots under N deficient conditions.2.Four GmNLA members were identified in soybean genome,which was named after GmNLA1,GmNLA2,GmNLA3 and GmNLA4,respectively.Among them,GmNLA1,GmNLA2 and GmNLA4 have relatively high similaritity with At NLA1,as reflected by more than 56% identity.Subcellular localization analysis showed that both GmNLA1 and GmNLA4 were located in the nucleus and cell membrane,while GmNLA2 was located only in the cell membrane.Histochemical localization analysis of their promoter fusion with GUS in soybean hairy roots showed that GmNLA1 and GmNLA2 were mainly expressed in the taproots,lateral root tips and lateral roots.However,GmNLA4 was mainly expressed in the taproots and lateral roots,suggesting that their biological functions may be different.3.The spatital and temportal expression patterns of GmNLAs members were regulated by nitrogen and phosphorus.Low nitrogen availability reduced the expression levles of GmNLA1/2/4 in young leaves under sufficient Pi conditions.Among them,GmNLA1 and GmNLA4 were down-regulated only after 7 d of N deficiency.Transcripts of GmNLA2 were down-regulated after 7 and 14 d of low nitrogen supplement.In roots,although GmNLA1/2/4 was down-reulated by N deficiency,GmNLA1 was observed at 14 and 21 d,while GmNLA2 at 14 d,and GmNLA4 at 21 d.Under sufficient N conditions,compared to that at high Pi levels,the expression levels of GmNLA1 and GmNLA4 in young leaves were significantly decreased at 7 d and transcripts of GmNLA2 were significantly decreased at 21 d under low Pi conditions.In roots,the expression levels of GmNLA2 were significantly decreased at 7 and 21 d by P deficiency,GmNLA1 and GmNLA4 were observed at 21 d.4.Functions of GmNLA1/2/4 were further examined in soybean hairy roots with GmNLA1,GmNLA2 or GmNLA4 RNA interference and overexpression.The results showed that GmNLA2 and GmNLA4 RNA interference significantly increased total phosphorus concentration in soybean hair roots under high or low phosphorus with sufficient N conditions except for GmNLA1 under low phosphorus conditions.Furthermore,it was observed that increasing total P concentrations was higher in transgenic lines with GmNLA1/2/4 compared with CK under high or low nitrogen with sufficient P conditions.However GmNLA1/2/4 overexpression had no effect on total P concentration in soybean hair roots.In summary,the molecular mechanism underlying GmNLA1/2/4 regulating of phosphorus balance under nitrogen stress was eluciated,which lays the foundation for further understanding of coordinated adaptation of soybean to nitrogen and phophorus deficiencies,and supplies the candidate gene and materials for development of soybean varieties with both high nitrogen and phosphorus efficiency.