Cloning And Functional Characterization Of Maize ZmNLP Genes In Nitrate Signaling

Nitrogen is one of the most critical macronutrients for plant growth and development.Nitrogen in the soil that can be absorbed by plants,and the forms included are nitrate and ammoniumn,most plants that grow in aerobic soils absorb nitrogen mainly in the form of nitrate.In agriculture,nitrogen fertilizer has been widely used to maintain the high yield of crops,but nitrate in soils cannot be completely absorbed by plants.Therefore,a large part is lost to the environment,resulting in serious environmental pollution such as acidification of the soil,eutrophication of water bodies and air pollution,affecting the survival and development of mankind.To solve these problems,it is right to improve the nitrogen use efficiency(NUE)of plants.Maize demand a large amount of nitrogen,but researches on nitrate regulatory genes in maize are still poorly understood.Thus,identification and characterization of nitrate regulatory genes and underlying mechanisms in maize is of great importance,especially in crop plants.AtNLP7(NIN-like protein 7)is an essential nitrate regulated gene in Arabidopsis.In this study,we identified nine ZmNLP genes using bioinformatics,and named them according to their location on chromosome.Phylogenetic tree based on the full length amino acid sequences of different species were revealed,which illustrates three conserved domains.We analyzed the expression profiles of ZmNLP genes and found that the expression levels of ZmNLP6 and ZmNLP8 were higher at different stages and different tissues and ZmNLP4 and ZmNL9 exhibited the lowest expression levels.Moreover,most ZmNLP genes were not induced by low and high nitrate concentrations.We cloned ZmNLP6 and ZmNLP8 and transformed them into Arabidopsis nlp7-4 mutant.The nlp7-4 plants contain NRP::YFP construct and are defective in nitrate regulation of nitrate responsive genes.The YFP fluorescence of transgenic lines can restore to wild type levels.And the expression of nitrate responsive genes NIA1,NiR and NRT2.1 in transgenic lines can be restored to the wild type level after nitrate induced.These results indicate that ZmNLP6 and ZmNLP8 are nitrate regulated genes.The nitrate content,nitrate reductase activity,and amino acid content in transgenic lines can recover to the wild type level,indicating that ZmNLP6 and ZmNLP8 can affect nitrate assimilation.The GFP fusion proteins are transiently transformed into Nicotiana benthamiana epidermal cells and Arabidopsis mesophyll protoplasts,showing that Zm NLP6 and ZmNLP8 are localized in the nucleus.From yeast-one hybrid,we can see that ZmNLP6 and ZmNLP8 proteins can directly bind nitrate-responsive cis-element(NRE)of ZmNRT1.2 and ZmNiR2 in vitro.The phenotypic analysis of the transgenic lines shows that ZmNLP6 and ZmNLP8 can increase the growth of the shoots and shoots under both low and high nitrate concentrations and increase the yield per plants under low nitrate concentrations.In this study,we found ZmNLP6 and ZmNLP8 play essential roles in nitrate signaling and assimilation.So far,this is the first detected nitrate regulatory genes in maize.These results provide the possibility for the breeding of new nitrogen-efficient maize varieties.