Transport And Absorption Of AuNPs Of Different Sizes In Tea Plants And Their Effects On The Metabolism Of Tea Suspension Cells

With the development of nanotechnology,nanoparticles(NPs)have been used in various fields.In agriculture,a wide range of nanoproducts such as nanoterres,nano-herbicides,nano-fungicides and nanopesticides can be used to improve plant growth and productivity.However,the widespread use of nanomaterials in plant production results in the release of a large amount of nanomaterials into the environment.However,the use of nanomaterials can also lead to new toxic effects on plants,comparable to those of traditional fertilizers,pesticides and plant protection products.Plants absorb nanomaterials of different sizes and penetrate them in different ways.Gold nanoparticles(AuNPs)are of great interest because of their electrical,mechanical,thermal,chemical and optical properties,and gold nanoparticles are still dominant and outstanding compared to other metal nanoparticles.The synthesis of AuNPs of different sizes and their use as a model to study the effects of their uptake,transport,and metabolism on tea tree suspension cells are of great importance for the study of the uptake and transport properties of different size nanomaterials in the tea tree body and for the evaluation of the toxicity of different size nanomaterials to plant cells.The main studies are:Four AuNPs with different sizes(5 nm,15 nm,50 nm,100 nm)were synthesized by tannic acid method,sodium borohydride method and citrate method,respectively.The small-sized AuNPs(<10 nm)synthesized by the tannic acid and sodium borohydride methods were compared with TEM and the experimental results showed that the AuNPs from the tannic acid method were nearly circular,uniform in size and better dispersed.The15 nm,50 nm,and 100 nm AuNPs synthesized by the citrate method were characterized by TEM and showed almost circular shape,uniform size,and good dispersion.The AuNPs of different sizes had different colors,with 5 nm AuNPs being orange,15 nm AuNPs redorange,50 nm AuNPs red-wine,and 100 nm AuNPs red-violet.The absorption shifts of the synthesized AuNPs were 510 nm,520 nm,530 nm,and 540 nm,respectively,which matched the range of absorption shifts of AuNPs and the blue and red shift patterns of SPR peaks measured by UV-Vis irradiation for AuNPs of different sizes.2.In the present study,four sizes of synthetic nanoclays(5 nm,15 nm,50 nm,and 100nm)were used to study the uptake and displacement of nanoclays of different sizes in tea plants using root and leaf application methods.1.5 ml of the studied nanoclay was added to the nutrient solution of hydroponic tea plants and sampled after 6 h,1 d,3 d and 5 d.The samples were washed and divided into three parts: Roots,Stems and Leaves.The Au content of each part was determined by ICP-OES at different time points during the treatment,and the results of the study showed that Au was detected in all parts of the control and treated groups,with the highest Au content in the leaves.The Au concentrations in the roots of tea plants treated with AuNPs of different sizes were higher than those of the blind control,indicating that tea plant roots can absorb AuNPs of 5 to 100 nm and accumulate them in the roots.200 μl of nanobeads of different sizes were applied dropwise to the abaxial surface of young leaves of hydroponically grown tea plants,and samples were taken 6 h,1 d,3 d,and 5 d after application.The samples were washed and divided into four parts: old leaves,young leaves,roots,and stems,and the Au content of each part was determined by ICP-OES at different time points during treatment.The Au content of young leaves increased significantly after treatment.This indicates that AuNPs in the range of 5-100 nm can be absorbed in young tea leaves.The experimental results showed that AuNPs of different sizes can be absorbed by the abaxial surface of tea roots and young leaves.3)In accordance with previous group studies,sterile tea seeds and sterile leaves differentiated from axillary buds of branches collected in the field were used to induce guarain tissue,and free guarain tissue was obtained,cut and inoculated into B5 culture medium,and succussed every 20 days to obtain suspended cells.Suspended cells of equal length and comparable viability were selected and supplemented with 1 ml of four different sizes of Au NP,while the control group was supplemented with ultrapure water.The suspension cells rapidly lost viability after the addition of 5 nm and 15 nm AuNPs.The viability of suspension cells treated with 50 nm Au NP gradually decreased with increasing number of days of treatment.After 30 days,all cells had lost viability,whereas half of the cells in the group treated with 100 nm AuNPs remained viable after 30 days.Based on the experimental results,the small size AuNPs(5 nm,15 nm)were more toxic to tea tree suspension cells,while the addition of 50 nm and 100 nm AuNPs was less toxic to the cells in the short term.This study provides insight into the future use of nanomaterials of different sizes as nanopesticides,etc.on tea trees and their effects on tea trees.Therefore,in the following experiments,suspension cells treated with larger size AuNPs(50 nm and100 nm)were selected for a non-targeted metabolomics assay to study the effect of different size AuNPs on the metabolic pathways of tea tree suspension cells.4.Untargeted metabolomic analysis of suspension cells treated with 50 nm and 100 nm AuNPs by LC-QTOF/MS.VIP values of P and OPLS-DA models were verified by statistical analysis.84 different metabolites(12 in positive ion mode and 62 in negative ion mode)between 50 nm and 100 nm AuNPs and blank control;26 different metabolites(3 in positive ion mode and 23 in negative ion mode)between 50 nm and 100 nm AuNPs treated groups.The metabolic effects on suspension cells after treatment with AuNPs of different sizes were mainly related to amino acid synthesis.Between the groups treated with 50 nm and 100 nm AuNPs and the control group,the following metabolic pathways were significantly affected: phenylalanine metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,arginine biosynthesis,etc.The following metabolic pathways were significantly affected between the 50 nm AuNPs and 100 nm AuNPs treatment groups:Vitamin B6 metabolism.The main metabolic pathways significantly affected between the50 nm AuNPs and 100 nm AuNPs treatment groups were vitamin B6 metabolism,arginine and proline metabolism and aminyl-tRNA biosynthesis.The experimental results of ICP-OES determination of the change of Au content in tea plant culture medium and on the back of young leaves of tea plants showed that AuNPs of5-100 nm could be absorbed into tea plants through the roots of tea plants and the back of young leaves of tea plants.Combined with KEGG metabolic pathway analysis and TTC method to determine the change of cell viability,after 50 nm AuNPs treatment,the metabolic pathway of cells is more perturbed,and the speed of cell loss of vitality is faster,and it is speculated that the use of nanomaterials with a size of 100 nm as a carrier can not only be absorbed by tea plants,but also have less metabolic interference and toxicity to tea plants.