Effects Of Nitrogen Concentration On Tomato Seedling Growth And Phenylpropane Biosynthesis Pathway

Nitrogen is the material basis of life activities,and the change of nitrogen level significantly affects plant growth and the production of secondary metabolites.In the process of tomato planting,farmers blindly applied nitrogen fertilizer in pursuit of high yields,resulting in changes in the content of secondary metabolites,which ultimately led to the decline in disease resistance and tomato quality.Phenylpropane biosynthesis pathway is an important secondary metabolic pathway to plant body weight,which can eventually to produce phenols,flavonoids and alkaloids involved in plant defense response,so that plants have certain resistance to the external environment.In this study,’Micro Tom’tomato was used as experimental material,and five nitrogen concentrations（0.384 mmol/L,1.534 mmol/L,7.670 mmol/L,15.340 mmol/L,23.010mmol/L）were set.We measure the growth indexes,photosynthetic indexes and antioxidant indexes of tomato seedlings.Screening of phenylpropane biosynthesis pathway differential genes to perform gene cloning,physicochemical property analysis and relative expression detection,and determination of key enzyme activities and product content of this pathway,we identified the family members of a key gene of phenylpropane biosynthesis pathways,and perform bioinformatics analysis,determination the gene expression level and construction of overexpression vectors for subcellular localization of the identified key genes of the phenylpropane biosynthesis pathway.The main results are as follows:1.The effect of different nitrogen concentrations on the growth of tomato leaves were 15.340 mmol/L>7.670 mmol/L>1.534mmol/L>23.010 mmol/L>0.384 mmol/L,The effect on photosynthesis was basically the same as that on growth,that is,the nitrogen concentration of 15.340 mmol/L was the most suitable concentration for the growth of tomato seedlings.Both high and low nitrogen concentrations affected the plant antioxidant system,malondialdehyde（MDA）,proline（Pro）,hydrogen peroxide（H₂O₂）and superoxide anion（O₂^-）were significantly changed.High nitrogen concentrations enhanced the activities of POD and CAT,indicating that when nitrogen concentration was too high（15.340 mmol/L、23.010 mmol/L）,tomato seedlings were subjected to a certain degree of stress,and promoted the activity of antioxidant enzymes to enhance and interact with each other to resist stress.2.A total of 51 differential genes of the phenylpropane biosynthesis pathway were identified from the transcriptome of our laboratory.There were 16 POD genes,10 Bgl genes,2 CYP98A genes,4 CAD genes,6Co AMT genes,1 CCR gene,7 PAL genes,2 HCT genes,2 4CL genes and1 CYP73A genes,respectively.Most of the genes had the highest expression when nitrogen concentration was 15.340 mmol/L.The key genes POD5,Bgl9,CYP98A1,CAD2,Co AMT1,PAL2,4CL2 and HCT2were screened for fluorescence quantification,and it was found that the expression of all eight genes changed significantly with time goes under nitrogen treatment,except for Bgl9 and PAL2.The expression levels of the other six genes reached the highest at 15.340 mmol/L of nitrogen treatment on the 9th day.The response of 4CL gene to nitrogen concentration was high nitrogen>control>low nitrogen,but the expression of PAL gene was reduced at high nitrogen concentration.Both high and low nitrogen concentration treatments enhanced the activity of 4CL enzyme and PAL enzyme,and the activity of 4CL enzyme was more sensitive and responsive to nitrogen than PAL.Both high（23.010 mmol/L）and low nitrogen（0.384 mmol/L）concentration treatments enhanced 4CL enzyme activity and PAL enzyme activity,and 4CL enzyme activity was more sensitive and responsive to nitrogen than PAL.and the contents of lignin and flavonoids increased with the extension of nitrogen treatment time.The content of lignin was different at the early stage of nitrogen treatment,but there was little difference at the late stage.At the early stage,the content of flavonoids was lower at 0.384 mmol/L nitrogen treatment than that of the other four treatments,and lower than that of the normal nitrogen treatment in the whole process.At the late stage,high nitrogen concentration（15.340 mmol/L,23.010 mmol/L）inhibited the synthesis of flavonoids.Tomato seedlings were subjected to nutrient stress at different nitrogen concentrations,especially at 0.384 mmol/L and23.010 mmol/L,which caused great damage to plant.However,the contents of lignin,polyphenols and flavonoids were higher at 15.340mmol/L nitrogen treatment.3.Six members of the tomato 4CL gene family were identified,encoding amino acids between 538-957,Only Sl4CL05 was a basic amino acid,all members were stable proteins and had no signal peptide,the subcellular localization predicted that all 6 proteins were expressed in peroxisomes,Sl4CL01 can also express in chloroplasts.The number of introns in tomato 4CL gene family ranged from 5 to 11,and the 12 motifs were highly conserved in sequence and location in Sl4CL,with high homology.Analysis of promoter cis-acting elements revealed that all members had a high number of light-responsive elements.The expression of Sl4CL01,Sl4CL03,Sl4CL04 and Sl4CL05 were highest at 15.340mmol/L after different nitrogen concentrations treatment.The Sl4CL03overexpression vector was constructed,and the subcellular localization results showed that it could be expressed in peroxisomes,chloroplasts and cytoplasm.