| Corn is one of the major food,feed and energy crops in the world.Organic phosphorus is a major component of phosphorus in soil,and can not be absorbed or utilized directly by plant.The soil phosphorus is not sufficient for plant growth and development,and phosphorus deficiency will seriously affect the yield and quality of maize.Therefore,the study on the mechanism of resistance to low phosphorus is significant in breeding strategies for low phosphorus.With the development and application of sequencing technology and functional genomics,many genes responding to low phosphorus stress have been identified in maize.However,few researches have been done on the function of these genes at present.Purple acid phosphatases(PAPs),a kind of metalloprotease,have been identified and characterized from numerous plants.These enzymes catalyze the hydrolysis of a wide range of organophosphorus substrates.The biological roles of this family are diverse in plants,including phosphate acquisition,phosphorus metabolism and transport during leaf senescence,disease resistance,abiotic stress resistance and so on.A characteristic sequence pattern with five highly conserved motifs(GDXG,GDXXY,GNHE/D,VXXH and GHXH,where X indicates any amino acid)has been identified in known and predicted PAPs.Within these five motifs,the seven invariant residues shown in bold represent the ligands that coordinate directly to the metal ions in the active site.In this study,PAPs were identified through genome-wide screening in maize inbred line B73.Nineteen ZmPAPs containing the five conserved motifs were identified in maize,and were named based on the highest homologous AtPAPs.q-PCR analysis revealed that NaCl,H2O2 and ABA stresses caused a general induction of ZmPAP7c expression in shoots of water cultured maize seedling,phosphorus(Pi)starvation induced the expression of ZmPAP13 and ZmPAP21c in roots and shoots and ZmPAP7d expression was induced by phosphorus(Pi)starvation,NaCl,H2O2 and ABA stresses.ZmPAP13 was located in the nucleus of maize protoplast.ZmPAP13 protein from prokaryotic expression system were functional in catalyzing hydrolytic reactions.Ectopic expression of ZmPAP13 in transgenic Arabidopsis thaliana increased the phosphatase activity of root surface,indicating that ZmPAP13 secretes to the root surface.In the condition of ADP or phytate as the sole P source,the weight of ZmPAP13 transgenic Arabidopsis thaliana was significantly higher than that in wild-type control.The physiological roles of ZmPAP13 is associated with increasing the utilization soil organophosphorus,thus ZmPAP13 plays an important role in plant tolerance to Pi limitation.Further analysis on the regulation of ZmPAP13 revealed that ZmPHR1 could activate the ZmPAP13 promoter activity.The secretion to the root surface of ZmPAP7d fused with the N signal sequence of ZmPAP13 manifested that the N signal sequence is crucial for the secretion characteristic of ZmPAP 13.Total phosphatase activity of Arabidopsis thaliana plants transformed with the construct of ZmPAP13 without the N signal peptide showed no different with that in wide type,confirming that N signal sequence is essential for the enzymatically active of ZmPAP13 in plants.Deletion of the N signal sequence resulted in aggregation of the ZmPAP 13 protein in maize protoplasts.Acid phosphatase activity and peroxidase activity was observed in ZmPAP7d protein from prokaryotic expression system,and ZmPAP7d could not secrete to the root surface.Under Pi starvation condition,no significant differences in seedling growth were observed between the transgenic Arabidopsis thaliana plants and the wide type.These results indicated that ZmPAP7d could not improve tolerance to Pi limitation.Heterologous expression of ZmPAP7d in Arabidopsis enhanced the tolerance to NaCl and mannitol stresses.AtPAP17 is highly homologous with ZmPAP7d.atp ap17,T-DNA mutation of AtPAP17,had lower seed germination rates than that in wide type under NaCl and mannitol stresses.These results suggested that ZmPAP7d plays a significant role in responding to osmotic stress.To further confirm the functions of ZmPAP 13 and ZmPAP7d,the overexpressed vectors of ZmPAP13 and ZmPAP7d were transformed into maize and the transgenic maize plants were obtained.Taken together,ZmPAP13,secreted to the root surface,is responsive to low phosphorus stress and involved in improving the utilization soil organophosphorus.ZmPAP7d,induced by NaCl,H2O2 and ABA stresses,is a member of the factors in the pathways to response to osmotic stress. |
PDF Full Text Request