Studies On The Interaction Between Nitrate Transport And NO In Response To Cadmium In Fraxinus Mandshurica And Betula Platyphylla

Nitrogen is an essential nutrient for plant growth.Nitrate uptake,as the source of nitrogen assimilation,plays an important role in regulating plant growth and responding to stress.As a nitrogen metabolites,the mutual regulation of nitric oxide（NO）and nitrate is not clear.Fraxinus mandshurica and Betula platyphylla are the main tree species in northeast China.However,there is no research on nitrogen assimilation of these two species under adverse conditions,especially under cadmium pollution.Therefore,this paper investigated the effects of NO and cadmium stress on nitrate uptake and assimilation of Fraxinus mandshurica and Betula platyphylla.The main contents are as follows:Based on the transcriptome data of Fraxinus mandshurica,37 FmNRT family members were identified,including 33 FmNRT1 and 4 FmNRT2.Based on the genome data of Betula platyphylla,47 BpNRT family members were identified,including 44 BpNRT1 and 3 BpNRT2.Compared conserved motifs of FmNRT and BpNRT,showed that there were significant differences betweenNRT1 andNRT2 in conserved motifs,but there was little difference between different species in the same subfamily.2.5mmol/L nitrate treatment（24h）decreased the synthesis and metabolite accumulation of NO in Fraxinus mandshurica and Betula platyphylla seedlings,while nitrate treatment with25mmol/L showed an increasing trend.Taking Fraxinus mandshurica as an example,under2.5mmol/L nitrate treatment,the activities of nitrate reductase（NR）and nitric oxide synthase（NOS）,the key enzymes for NO synthesis,were 18%and 1.3%lower than control group respectively.The contents of NO and its metabolite,nitrosothiol（SNO）,were 58%and 36.6%lower than control group respectively,while the activity of nitrosoglutathione reductase（GSNOR）,a key enzyme that catalyzes the regulation of NO and SNO levels,was 25%higher than control group;The results of 25mmol/L nitrate treatment were opposite to the above treatment,SNO content increased the most,was 72%higher than control group.It can be seen that nitrate has concentration effect on NO synthesis and accumulation.1mmol/L NO donor sodium nitroprusside（SNP）promoted the NO₃^-uptake and assimilation in Fraxinus mandshurica and Betula platyphylla,while the 80μmol/L GSNOR inhibitor（N6022）decreased the NO₃^-uptake and assimilation.Taking Fraxinus mandshurica as an example,under NO treatment,the expression of NO₃^-transporter genes FmNRT1.1 and1.2 were significantly increased,were 115%and 124%higher than control group respectively,NO₃^-content was 13%higher than control group.The contents of NO₂^-,NH₄⁺and Glu were22%,31%and 41%higher than control group respectively.The activities of NR,nitrite reductase（NiR）and glutamine synthase（GS）,which were the key enzymes of NO₃^-assimilation,were 20%,33%and 35%higher than control group respectively.The results of SNO treatment were opposite to NO treatment,among which FmNRT2.1 gene expression and Glu content were 98%and 72%lower than control group respectively.Therefore,NO and its metabolite SNO can change NO₃^-uptake and assimilation.Fraxinus mandshurica and Betula platyphylla seedlings were treated with cadmium chloride at different concentrations（0,10 and 50mmol/L）,The growth of Fraxinus mandshurica was promoted by 10mmol/L Cd²⁺treatment,and inhibited by 50mmol/L Cd²⁺treatment.The growth of Betula platyphylla was inhibited by Cd²⁺treatment,and the inhibition increased with the increase of Cd²⁺concentration.The growth of Fraxinus mandshurica and Betula platyphylla were inhibited by 50mmol/L Cd²⁺treatment（30d）,and the inhibition was alleviated by NO or nitrate.Cd²⁺treatment promoted the synthesis and metabolite of NO.In addition to the increase of NO₃^-uptake in Betula platyphylla,the NO₃^-metabolism in Fraxinus mandshurica and Betula platyphylla was inhibited by Cd²⁺treatment,and its inhibition was alleviated by NO.In addition,Cd²⁺treatment increased the NH₄⁺content of Fraxinus mandshurica,but decreased the NH₄⁺content of Betula platyphylla.It can be seen that NO synthesis and metabolite of Fraxinus mandshurica and Betula platyphylla have the same response trend to Cd²⁺treatment,but the uptake and assimilation of NO₃^-are different.Analysis of transcriptome data of Fraxinus mandshurica roots treated with 50mmol/L Cd²⁺for 30 days showed that the expression of high-affinity NH₄⁺transporter Fm AMT1.1 gene was 240%higher than control group.while the expression of the NO₃^-transporter FmNRT2.1gene was 72%lower than control group.At the same time,it was found that the soluble sugar content was 24%lower than control group,and the C/N was 82%higher than control group.The above results further confirmed that Cd²⁺treatment decreased NO₃^-uptake and increased NH₄⁺uptake,which may be due to the change of transporter activity.In order to analyze the effects of FmNRT2.1 on NO synthesis and metabolite,In Fraxinus mandshurica with transient silencing of FmNRT2.1,the expression of Fm NIA decreased by36%,the expression of Fm NOS increased by 183%,and the contents of NO and SNO decreased by 18%and 23%,and the GSNOR activity decreased by 29%compared with the control group,It can be seen that the level of NO synthesis and metabolism is inhibited by FmNRT2.1 silencing,and this inhibition can be alleviated by the application of nitrate.