The Functional Characterization Of BnaC2.TPS8 In Response To Nitrogen Starvation In Brassica Napus

Nitrogen is essential macronutrients for plant growth.Nitrogen deficiencycould inhibited the metabolic activity in plants,and lead to the accumulation of carbohydrate,and the imbalance of carbon and nitrogen nutrients in plants.The Class I TPS genes in crops involoved in the development of plants and the responses to abiotic stress,such as drought,salt,low N and high sugar.However,the function of the Class II TPS genes are not clear.Oilseed rape(Brassica napus L.)is an important oil crop in China as well as in the world,which needs more nitrogen than other species.Bna C2.TPS8 is one of the Class II TPS genesin Brassica napus.In this study,the overexpression lines of Bna C2.TPS8 in Arabidopsis and Brassica napus and the CRISPR-Cas line of Bna C2.TPS8 in Brassica napus were used to study the physiogical function and molecular mechanism of Bna C2.TPS8,which will provide the theoretical basis in the balance of carbon and nitrogen metabolism in Brassica napus.Shoot and root growth;the concentration of anthocyanin,chlorophyll,sucrose and starch in leaves;the content of total carbon and nitrogen nutrients,the ratio of carbon to nitrogen;the activity of NR,GS and other enzymes;the gene expression profile related to anthocyanin synthesis,sugar metabolism,nitrogen uptake,transport and assimilation in wild type,Bna C2.TPS8 overexpressed lines in Arabidopsis and Brassica napus and and CRISPR-Cas lines in Brassica napus were investigated under different N and sugar conditions by hydroponic culture and agar culture.The main results are as follows.(1)Compared with the wild type,Arabidopsis Bna C2.TPS8 overexpression lines showed more sensitive to nitrogen deficiency and high sugar growth conditions.There were significant differences in the biochemical and physiological parameters between Col-0 and Bna C2.TPS8 overexpression lines OE-2,OE-7 under optimal nitrogen(30 m M N)and low nitrogen(0.3m M N)conditions by hydroponic culture.Compared with the wild type,the color of leaves got darker,the number of leaves decreased in Arabidopsis Bna C2.TPS8 overexpression lines under optimal N conditions.However,there were no difference in the shoot and root fresh weight between wild type and Arabidopsis Bna C2.TPS8 overexpressed lines.Compared with the wild type,Arabidopsis Bna C2.TPS8 overexpression lines showed a more sensitive phenotype to nitrogen deficiency,for example,the number of leaves,the biomass,the Fv/Fm and the chlorophyll concentration decreased significantly;anthocyanin and malondialdehydeconcentration increased significantly;the concentration of sucrose and starch in the bottom leaf(purple)increased significantly;total nitrogen and total carbon concentration decreased and increased significantly respectively,the ratio of carbon to nitrogen increased significantly.The more serious inhibition of plant growth in Arabidopsis Bna C2.TPS8 overexpression lines under low N could be attributed to the inhibition of energy metabolism caused by the imbalance of C and N under the environment of low nitrogen and high carbon for a long timeUnder the treatment of 1% Glc,the germination rate and the greening rate of cotyledons of Bna C2.TPS8 overexpression lines reducded more than that of the wild type.Moreover,the leaves of the Bna C2.TPS8 overexpression lines had more anthocyanin treated with low N and high sugar for a long period,whether under long day or short day conditions;and lower Fv/Fm under long day condition.(2)As compared with the wild type,the Brassica napus Bna C2.TPS8 overexpression lines showed more sensitive to N deficiency.Under the optimal N treatment,compared with the wild type(ZS11),the biomass of the Bna C2.TPS8 overexpression lines increased significantly,the chlorophyll concentration of L1 and L2 decreased and increased significantly,respectively;the expression amount of Bn PPT(phosphoenolpyruvate translocator),Bna C3.GLT1(Glucose-6-phosphate translocator 1)and Bn LHCII(light harvesting complex II)were increased,and that of Bna C.GLN1a(Glutamine synthetase 1)and Bna A03.SGR(Bna A03.SGR,Stay green)were decreased in the leaf.Under the nitrogen deficient treatment,compared with the wild type,L1 and L2 of the Brassica napus Bna C2.TPS8 overexpression lines became more purple;the anthocyanin,chlorophyll and malondialdehyde concentrations in leaves,andsucrose and starch concentration of L2 in the Bna C2.TPS8 overexpression lines were were all significantly increased;the relative expression amount of genes related to anthocyanin synthesis,Bna-N-INVB(Sucrose invertase)and Bn SUC2(sucrose transporter 2)in leaves and Bna Cn.NRT1.7(Nitrate transporter 1.7)in roots in the Bna C2.TPS8 overexpression lines was significantly increased,whereas,Bn Susy1(sucrose synthase1)was significantly decreased.The responses of Brassica napus Bna C2.TPS8 CRISPR-Cas lines were on the contrary to the Brassica napus Bna C2.TPS8 overexpressing lines.Compared with the wild type,the biomass of Bna C2.TPS8 CRISPR-Cas lines were significantly reduced inoptimal nitrogen treatment;the leaves showed less yellow,the anthocyanin and malondialdehyde concentrations of L1 and L2 were significantly reduced,and the chlorophyll concentration was significantly increased in nitrogen deficient treatment.These showed that Brassica napus Bna C2.TPS8 overexpression lines showed much more sensitive than Brassica napus Bna C2.TPS8 CRISPR-Cas linesin response to nitrogen deficiency.