Physiological Characteristics And Key Gene Screening Of Nitrate Accumulation In Spinach

Spinach is an important leaf vegetable,however,the excessive accumulation of nitrate in spinach is not only harmful to human health,but also limits the improvement of nitrogen use efficiency.Therefore,it is necessary to reduce nitrate accumulation in spinach.Many researches have been conducted on the plant nitrate accumulation,especially at the molecular level.The present study compared the physiological and molecular characteristics of nitrate uptake,translocation and assimilation between 2-4 spinach genotypes with high or low nitrate concentration in hydroponic experiment,aiming to screen the key factors causing the different nitrate level between spinach genotypes.In addition,to explain the molecular mechanism of environmental factors effecting on nitrate accumulation in spinach,we analyzed the expression pattern of genes encoding SoNRTs and nitrogen assimilation enzymes with glutamine,high temperature and low light treatment.Finally,we cloned the full length CDS of 4 SoNRTs and analyzed the basic information of them by bioinformatics approaches.The specific results are as follows:1.A hydroponic experiment was conducted to study the difference of nitrate accumulation,uptake and utilization efficiency between four spinach genotypes.Results showed that S13 had the highest nitrate contents under two N level conditions,while S10 had the lowest nitrate contents.S18 had the similar nitrate contents with S13 under low N level,while it showed no significant difference of nitrate contents with S57 under high N treatment.The 15NO₃^--N uptake efficiency of S13 was the greatest one among four genotypes,while its N utilization efficiency and N utilization ratio were significantly lower than those of S18 and S57;The shoot dry mass,nitrate reductase activity,N utilization efficiency,N utilization ratio of S18 and S57 were greater than those of S13 and S10,while their 15NO3--N uptake rates were lower than those of S13.The shoot dry mass,nitrate reductase activity,NutE,N utilization ratio of S10 were significantly lower than those of S18 and S57,and its 15 NO₃^--N uptake rate was significantly lower than those of S13.Among the four spinach genotypes,the S57 can be selected as elite germplasm using for spinach production for its relatively lower nitrate content and higher N efficiency.2.A hydroponic experiment was conducted to study the transcriptional patterns of gene coding nitrate transporters and nitrogen assimilation enzymes.Results showed that the SoNRT1.1a/b,SoNRT1.5a/b/c,SoNRT2,SoNIA and SoGS1 were mainly expressed in root,while SoNRT1.4,SoNRT1.6,SoNRT1.9a/b and SoGS2 were mainly expressed in blade and petiole.The SoNRT1.2 and SoNRT1.3 showed no obvious tissue specific expression pattern.Generally,the expression level of the tested gene in spinach S18 was higher than that of S10.Compared with low N treatment,the expression of most genes increased by several even dozens of times under high N treatment,while the expression of SoNRT1.1b,SoNRT1.5a and SoNRT1.5c gene in the leaf of S18 and SoNRT1.3 in the root of S10 increased by more than one thousand times,at the same time the expression of SoNRT1.4 gene in S18 increased by nearly ten thousand times.Moderate positively correlations were observed between the expression level of SoNRT1.1b,SoNRT1.2,SoNRT1.5c,SoNRT1.9b and the blade nitrate content,as well as the expression level of SoNRTs,SoNIA,SoGS1 and petiole nitrate content,which further confirmed that the nitrate content difference between S10 and S18 is resulted from both nitrate uptake,transport and assimilation factors.3.Expression of gene encoding SoNRT1.1a/b,SoNRT1.2,SoNRT1.3,SoNRT1.4,SoNRT1.6,SoNRT1.5c,SoNRT2,SoAS,SoNIA,SoNIR,SoGS1/2,SoNADH-GOGAT and SoFd-GOGAT was studied after 1 h and 24 h of Gln（70 mg/L）,heat（36 ℃）and low light（10 μmol/m²/s）treatments.The expression of most SoNRTs in root were found to be down-regulated under three treatments,while SoNRT1.2,SoNRT1.3,SoNRT1.4,SoNRT1.5c,SoNRT1.6 and SoNRT2 showed high transcription levels in the later period of different treatments in spinach roots.Most of the genes encoding enzymes responsible for N assimilation were found to be down-regulated under three treatments,expect the enhanced level under 1 h heat treatment and unchanged levels of SoFd-GOGAT,SoGS1 and SoNIA under 1 h Gln treatment.The expression of most nitrogen assimilation enzyme genes in the ground part of spinach were inhibited under most treatments,except for the unchanged expression level of SoFd-GOGAT,SoGS1 and So NIA under 1 h Gln treatment and the up-regulated expression of all the assimilation enzyme genes under 1 h heat treatment.4.In the present work,genes encoding SoNRT1.1a,SoNRT1.3,SoNRT1.5c and SoNRT1.9b were cloned.These SoNRT proteins were found hydrophobic and consistent with the distribution of hydrophobic regions,and they share similar conserved motifs with Arabidopsis NRT proteins.Subcellular localization prediction showed that most of the SoNRT proteins were distributed on the cell membrane.The molecular phylogenetic analysis showed that SoNRT1.1a and SoNRT1.5c were more similar to BvNRT1.1 and BvNRT1.5 proteins,respectively,which confirmed the close relationship between them.