Responses Of BpNPR1 Family Members And Triterpenoid Accumulation To Cadmium,Light And NO In Betula Platyphylla

Nonexpressor of pathogenesis-related genes 1（NPR1）is an important regulator of plant resistance and an acceptor of the signaling molecule salicylic acid（SA）.NPR1 enters the nucleus as a monomer and performs its function,while its conversion between oligomerization and monomerization is regulated by SA,the NO donor nitrosoglutathione（GSNO）,and key enzymes for denitrosylation,among others.Previous experiments have confirmed that SA promotes the synthesis of triterpenes such as betulinic acid and oleanolic acid in birch seedlings and healing tissues,but whether or how NPR1 is involved in triterpene synthesis remains unreported.Therefore,NPR1 family genes were hereby screened and identified in this thesis based on birch genomic and transcriptomic data;the ROS/RNS balance,NPR1 family gene expression and triterpene accumulation under light treatment,Cd treatment and NO treatment;NPR1 family members significantly associated with triterpene accumulation were screened;and their functions in triterpene synthesis were verified using transient transformation experiments.The main results are as follows.A total of four BpNPR1 family members were identified based on birch transcriptome and genome data,named BpNPR1,BpNPR2,BpNPR3,and BpNPR4 in accordance with their chromosomal localization,and were detected with amino acid lengths ranging from 354 to 605bp and localized on chromosomes 1,3,11,and 14,respectively.The promoter prediction analysis revealed that all of them were furnished with response elements such as SA,light,and abiotic stresses on them.After 10 days of light treatment of birch seedlings,chlorophyll a/b values were found not significantly different from the control,while the content of leaf dry weight,H₂O₂ content and its metabolic key enzyme activity,NO synthesis and metabolites,and triterpene content and its biosynthetic key enzymes showed an increasing trend,among which,compared with the control,the content of NO,its metabolite nitrosothiol（SNO）and triterpene content increased1.41,1.71 and 1.29 times,respectively.However,the BpNPR1 family genes responded differently to light treatment,with the highest expression of BpNPR2 gene being 3.57-time higher than that of the control.The results were contrary to the above findings after 10 days of dark treatment,where NO,SNO and triterpene contents were 0.69,0.25 and 0.77 times higher than the control,respectively;BpNPR2 gene expression was most significantly inhibited,0.71times that of the control.The treatments of 10 mmol/L and 50 mmol/L Cd²⁺inhibited the growth of Betula platyphylla,reduced the dry weight of leaves,and increased the content of H₂O₂,its metabolic key enzyme activity,NO synthesis and its metabolites,and triterpene content and its biosynthetic key enzymes,among which,compared with the control,the content of NO,its metabolite SNO,and triterpene increased 1.45,3.53 and 1.29 times,respectively.In contrast,the BpNPR1 family genes responded differently to Cd²⁺treatment,with the highest expression of BpNPR2 gene being 1.57-time higher than that of the control.The external application of NO and SA inhibited the production of H₂O₂,but further promoted the accumulation of NO and birch triterpenes,and up-regulated the expression of BpNPR2 genes to 2.92,1.28 and 3.64times that of the control,respectively.H₂O₂ content and its metabolic key enzyme activity,the content of NO,its metabolites and triterpene content,also its biosynthetic key enzymes presented an increasing trend after 1mmol/L NO donor sodium nitroprusside（SNP）treatment,among which,compared with the control,the content of NO,its metabolites SNO and triterpene increased 2.13,3.49 and 1.45times,respectively.In contrast,the BpNPR1 family genes responded differently to NO treatment,with the highest BpNPR2 gene expression being 2.86-time higher than that of the control.The above results were reversed after 1 mmol/L nitrate reductase inhibitor sodium tungstate（ST）treatment compared to NO treatment,when the NO,SNO and triterpene contents were 0.72,0.41 and 0.71 times that of the control,and the expression of BpNPR2genes was down-regulated to 0.76 times.The effect of NO on birch seedlings was attenuated by 1 mmol/L sulfhydryl reducer DTT,and the above results were not significantly different from those of the control.The BpNPR2 gene with a length of 1773 bp was cloned and transiently transformed into birch healing tissues.It was found that the overexpression of BpNPR2 gene significantly increased the expression of triterpene content and CAS2 gene,a key enzyme for its synthesis,20.3 and 80.46 times that of the control,respectively,while silencing of BpNPR2 gene significantly decreased the expression of triterpene content and its synthesis key enzyme gene;N6022 and Agrobacterium containing BpNPR2 gene overexpression vector were found to increase the triterpene content and its synthesis key enzyme gene of birch by 2.76～40.16 times compared with overexpression of BpNPR2 gene alone after simultaneous treatment of birch healing tissues;backfilling birch BpNPR2 gene into Atnpr1-1 mutant revealed a triterpene content 1.22 times that of the control,and a triterpene content 1.38 times that of the control in the Atnpr1-1 mutant treated with N6022 backfilled with the BpNPR2 gene than the BpNPR2Arabidopsis mutant overexpressed alone.