Molecular Mechanisms Of Brassinosteroid Regulation Of The Carbon And Nitrogen Balance In Plants

Plants use the products of photosynthesis and sugar metabolites glucose and sucrose as carbon(C)sources,and use inorganic nitrogen(nitrate nitrogen and ammonium nitrogen)as nitrogen(N)nutrients to maintain growth and development.In addition to the basic requirements as an energy source and structural composition,the balance of carbon and nitrogen nutrients plays a key regulatory role in the growth and development of plants.Brassinosteroid(BR)is an important plant sterol hormone,which plays an important regulatory role in plant sugar metabolism and nitrogen metabolism,but the molecular mechanism of BR in regulating plant carbon-nitrogen balance is still unclear.The protein kinase SnRK1 is an energy sensor in plants and plays a key regulatory role in the carbon-nitrogen balance of plants.In this study,molecular biology,biochemistry,cell biology,genetics and other methods were used to systematically study the molecular mechanism of BR and SnRK1 in the coordinated regulation of plant carbon/nitrogen balance.The specific research results are as follows:1.Brassinolide enhances the resistance of plants to the imbalance of carbon and nitrogen.Based on statistics of the green ratio of BR-related mutant seedlings grown on high-carbon/low-nitrogen and high-carbon/high-nitrogen media,it was found that the BR loss function mutant was more sensitive to the imbalance of carbon and nitrogen than the wild type,and showed it’s leaves more easy to turn purple,and the BR gain function mutant has slightly more green leaves than the wild type,indicating that BR enhances the stress resistance of plants to the imbalance of carbon/nitrogen ratio.2.BIN2 and BZR1 play a key role in BR regulation of carbon/nitrogen balance.Experimental studies have shown that the BR gain function mutant bzr1-1D can partially restore the stress resistance of BR synthetic deficient mutant det2-1 to carbon/nitrogen imbalance.At the same time,we found that BIN2,a key negative regulator in the BR signaling pathway,is essential for plants to respond to carbon/nitrogen imbalance.3.BIN2 interacts with KIN10 and phosphorylates each other.Our experimental results show that BIN2 can interact with the kinase subunit KIN 10 of plant-specific energy receptor SnRK1.In vitro kinase activity analysis showed that KIN 10 protein can phosphorylate BIN2,and BIN2 can also phosphorylate KIN 10,and the site where KIN10 is phosphorylated by BIN2 was identified by mass spectrometry.4.BIN2 enhances the activity of KIN 10.Protoplast transient transformation system detection showed that BIN2 could significantly enhance the regulation of KIN10 on downstream gene expression.The activity analysis of KIN 10 phosphorylation sites after mutation showed that KIN10 Thy175 and Ser179 were the most critical functional sites for BIN2 to regulate KIN 10.When these two sites were mutated,KIN 10 lost regulation of the downstream gene DIN6.5.KIN10 regulates A rabidopsis response to carbon/nitrogen imbalance.Comparing the greening ratio of KIN10-related mutant seedlings grown on a carbon/nitrogen imbalance medium(high carbon/low nitrogen;high carbon/high nitrogen),it was found that the KIN10 loss function mutant was more likely to turn purple than the wild type,and the KIN 10 gain function mutant was more able to maintain leaf greening than the wild type.It shows that KIN10 improves the stress resistance of plants to carbon/nitrogen imbalance.6.KIN10 is involved in BR-mediated regulation of carbon/nitrogen balance.When PPZ,the synthetic inhibitor of BR,was applied exogenously,the greening proportion of overexpressed KIN 10 seedlings remained at a high level,and the overexpressed KIN 10 plants were weakly sensitive to PPZ.The sensitivity of KIN10 mutant to BL was significantly lower than that of Col-0.In this study,it was found that brassinolide(BR)and SnRK1,the core element that regulates energy metabolism,synergically regulate the response of Arabidopsis to carbon/nitrogen imbalance and regulate the early growth and development of plants after germination,providing some new ideas for further studies on the responses of plants to key nutrient elements carbon and nitrogen.