The Influence And Acting Mechanism Of Flavonoids On Phosphorus Absorption And Utilization In Tobacco Plant

Phosphorus is involved in almost all material metabolism,energy metabolism,and cell life activities in plants.It also has a very important impact on crop yield and quality in agricultural production.Flavonoids are the most abundant polyhydric phenol secondary metabolites,which are ubiquitous in various plants.They not only affect the growth and development of plants,but also play an important biological role in the process of plants resisting stress.The flavonoids and their upstream and downstream biosynthetic metabolic pathways in most plants are strongly activated under P-deficiency situation,showing a significant increase in the content of flavonoids and upstream and downstream products.However,the mechanism of action of flavonoids in the homeostasis of plant phosphorus is unclear by now.In this study,the wild type of tobacco(common tobacco,HD)and a EMS mutant(Because its developed surface glandular hairs,and its dendritic shape,we named it Dendritic Non-Glandular trichomes,dng)were used for our research,through a series of physiological and biochemical,molecular biology and genome resequencing methods,preliminary exploration of the molecular regulation mechanism of flavonoids in the absorption and transport of plant phosphorus nutrients,and preliminary analysis of the DNG gene.Highlights of the present study are listed below:1.The results of phenotypic analysis of wild-type and mutant tobacco dng at different phosphorus supply levels show that the shoot,root architecture system and biomass in different tissue of dng mutants are not significantly different from wild-type tobacco at normal phosphorus supply levels;At P-deficiency situation,the main root of dng mutant was significantly shorter than that of wild type,and the main root length was only 48.6%of that of wild type,but the number of lateral roots and biomass in shoot and root were not significantly different from that of wild type tobacco.Compared with the wild type,at normal phosphorus supply conditions,there was no significant difference in the phosphorus content of the roots and roots of the dng mutant,while under P-deficiency supply conditions,the phosphorus content of the roots and roots of the dng mutant increased by 71.5%and 34.9%,respectively.These results indicate that the dng we obtained is a phosphorus-efficient tobacco mutant whose root development is regulated by systemic phosphorus signals.2.To explore the potential reasons for the difference in phosphorus efficiency between wild-type tobacco and mutant dng under P-deficient stress,we used LC-MS to perform a full-component test on wild-type HD and mutant dng at first step.Among the 210 kinds of chemical components detected,the content of flavonoids in dng was significantly lower than that of wild type.Therefore,we further conducted an accurate flavonoid targeted metabolomics analysis of wild-type and dng.The results showed that among the 62 flavonoids detected,the content of 27 flavonoids,including methoxy flavonoids,quercetin and chlorogenic acid were significantly reduced in the mutant dng,and no significantly increased flavonoids were detected in the dng,compared with wild-type tobacco.The above results suggest that the increase of phosphorus content in mutant dng at P-deficiency situation may be caused by the defect of flavonoid synthesis in the tobacco.Flavonoids can affect the absorption and utilization of phosphorus by plants.3.To verify how flavonoids specifically affect the absorption of phosphorus by plants,we treated wild-type tobacco with exogenous flavonoids under P-deficient conditions,and found that the content of flavonoids added with exogenous sources Increase,the phosphorus content in tobacco is significantly reduced,and the expression levels of multiple phosphate transporter gene NtPTs in response to phosphorus starvation signals are significantly up-regulated;and OsPT6::GUS transgenic tobacco materials that specifically respond to phosphorus deficiency signals are subjected to phosphorus deficiency conditions treatment with exogenous flavonoids,the results also found that compared with the control group without flavonoids,the addition of exogenous flavonoids significantly enhanced the GUS protein activity,indicating that the flavonoids under the condition of phosphorus deficiency exacerbated the plant’s phosphorus deficiency stress.Through the exogenous flavonoids replenishment experiment,we further detected the phosphorus content of the wild-type and dng mutant materials treated with flavonoids under phosphorus deficiency conditions.This result confirms that DNG-mediated flavonoids may act as a negative regulator in the absorption and transport of phosphorus by plants under phosphorus deficiency conditions,and its presence can inhibit the absorption and utilization of phosphorus by plants.4.Exogenous flavonoids negatively regulate the absorption and transport of phosphorus by plants,so we used the key rate-limiting enzyme chalcone synthase(CHS)that regulates the biosynthesis of flavonoids to study the effect of plant flavonoid synthesis on the absorption and transport of phosphorus.In order to verify whether CHS participates in the regulation of phosphorus nutrient absorption and translocation under phosphorus deficiency conditions,we first tested the response of tobacco NtCHS1 gene to phosphorus starvation signal under different phosphorus supply levels by qRT-PCR and promoter fusion GUS reporter gene method,It was found that the expression of NtCHS1 gene was strongly induced by phosphorus starvation signal,and the 1800bp NtCHS1 promoter could drive the downstream GUS reporter gene to significantly up-regulate the expression in P-deficient tobacco.We treated the transgenic tobacco material(CR)that specifically silenced by NtCHS1 with different phosphorus supply levels to further verify whether the changes in flavonoid levels in plants affect the steady-state balance process of phosphorus nutrients.The results found that under P-deficient conditions,compared with wild-type materials,the phosphorus content in the aboveground and belowground materials of CR materials increased significantly,indicating that NtCHS1 is strongly involved in the absorption and transportation of phosphorus nutrients under the condition of phosphorus deficiency,which can realize the regulation of the absorption and utilization of phosphorus by plants by regulating the flavonoid.5.The previous genetic studies of this research group showed that the mutation traits of dng mutants may be controlled by a recessive single gene.In this study,we used group separation analysis(BSA)and high-throughput genome resequencing technology to initially locate the mutant gene in the 6M interval of chromosome 13,which laid the foundation for further screening to locate the mutant gene and verify its function basis.In summary,we are convinced that flavonoids have a very critical role in the absorption and utilization of phosphorus nutrients by plants.We can reduce the level of flavonoids in plants through genetic engineering to improve the absorption efficiency of phosphorus nutrients by crops.we are convinced that flavonoids have a very important role in maintaining the homeostasis of tobacco phosphorus.DNG-mediated changes in flavonoids may negatively regulate the use of tobacco at P-deficiency.