The Uptake And Transportation Of Carbon Nanodots And Their Toxic Mechanism In Plants

As a novel fluorescent carbonic nanomaterial,carbon nanodots?C-dots?were recently discovered.They have several inherently unique characteristics,such as favorable water-solubility,facile modification,chemical inertness,excellent biocompatibility and low cytotoxicity.Based on all these advantages,the application of C-dots in biological imaginghas attracted more and more attention.Therefore,it has an important significance to assess the systemic toxicity of C-dots in organisms.However,the researches about biological toxicity of C-dots and their uptake were mostly focused on animals.As for plants,few studies regarding the effects of C-dots have been carried out,which severely constrained the application of C-dots in cellular imaging of plants.In this study,two plant species,maize?Zea mays L.?and Arabidopsis?A.thaliana?were chosen to assess the toxicity of carbon nanodots?C-dots?in plants.The C-dots were exposed to plants by hydroponics,sand culture and solid medium.Then the effects of C-dots on these plants were evaluated and the mechanisms were futher studied at both the physiological and molecular levels.Besides,by using the fluorescent microscopy,the uptake and transport of C-dots in maize andA.thaliana were also investigated.This study would establish scientific base for the application of C-dots in cellular imaging of plants.The main research methods and results were listed as follows:1.Synthesis and characterization of C-dotsC-dots used in this study were synthesized by one-step microwave synthesis route.After purification,the C-dots were futher characterizated by various methods.The morphology of the C-dots was characterized using a JEOL JEM-2100F high resolution transmission electron microscopy?HRTEM?,and the HRTEM image of C-dots showed that the size of C-dots was about 3 nm.The zeta potential of C-dots,estimated by a Zeta sizer Nano ZS,was-12.42±1.44 when dispersed in DI-water.By spectrophotometer,C-dots were found to have a broad absorption spectrum with peaks centered at 270 nm,340 nm,and 405 nm.And the emission fluorescence of C-dots was found to depend on the excitation fluorescence.The Raman scattering spectrum showed that the characteristic bands of C-dots were represented at around 1365 cm^-1 and 1615 cm^-1.Besides,FTIR spectroscopy showed that C-dotswere surrounded by O-H,N-H,C=O and CH₂.The stability of C-dots in DI-H₂O,1/2-strengthHoagland solution,and 1/2 MS was monitored by PL spectra.The fluorescence intensities remain stableduring the process of the experiment,whichsuggested no agglomerationoccurred.Therefore,C-dots were highly stable in these media.The spectrophotometeralso showed no differencebetween C-dots before and after sterilization.2.The uptake and translocation of C-dots in Maize and Arabidopsis thaliana.C-dots were observed in detached root-cap cells,cortex and vascular bundle of roots and mesophyll cells of leaves through fluorescence microscopy analysis,suggesting that C-dots were absorbed and translocated systemically in maize.Remarkably,a certain amount of C-dots were excreted out from leaf blade.And by fluorescence microscopy analysis,C-dots were also found to be absorbed and futher translocated to aerial tissuesinArabidopsis.3.The effects of C-dots in Maize and Arabidopsis thaliana.Firstly,the maize seeds of were exposed with C-dots at 0,250,500,1000 and 2000mg/L for 5 days.Root elongation of maize was significantly inhibited by C-dots at a high concentration of 2000 mg/L,while C-dots at 250-1000 mg/L showed no significant inhibition to mazie.After exposed in sand matrix amended with 0-2000 mg/L C-dots for 4 weeks,we found that 1000 mg/L and 2000 mg/L C-dots could significantly inhibit the grouth of maize seedlings.Conversely,activated carbon?AC?showed no toxicity to mazie even at a high concentration of 2000 mg/L.The effects of C-dots at 62.5-1000 mg/L on Arabidopsis thaliana?Arabidopsis?growth were evaluated after 7 days of exposure bysolid media.As compared to control and AC exposed plants,significant inhibition of plant growth was observed with 125-1000 mg/L C-dots exposure.Overall,the phytotoxicity of C-dots in plants was concentration-dependent.4.Thetoxic mechanism of C-dots in Maize and Arabidopsis thaliana.The exposure of C-dots at 2000 mg/L significantly increased the H₂O₂ content and lipid peroxidation in maize roots.Besides,the activities of antioxidant enzymes,including catalase?CAT?,ascorbate peroxidase?APOX?,guaiacol peroxidase?GPOX?and superoxide dismutase?SOD?,were also enhanced in varying degrees.Meanwhile,the exposure of C-dots at 1000 mg/L also significantly increased the H₂O₂ content in Arabidopsis roots.In addition,glutathione reductase?GR?activity was significantly up-regulated in roots and shoots,and the APOX activity was also significantly up-regulated in Arabidopsis shoots.In contrast to the control,the chlorophyll contents of the Arabidopsis seedlings after 1000 mg/L C-dots treatment were slightly decreased but the carotenoids contents were slightly increased.The activities of three important photosynthesis enzymes,including Rubisco,Fructose1,6-bisphosphatase?FBP?and Fructose 1,6-bisphosphate aldolase?FDA?were significantly inhibited by both 125 mg/L and 1000 mg/L C-dots treatment.GC-MS,as a highly efficient technique of metabolomics,was chosen in this study to reveal the metabolites changes in Arabidopsis seedlings.GC-MS separately detected and identified 48 and 71 metabolites in Arabidopsis roots and shoots,respectively.The identified metabolites were normalized and futher analyzed by Principal Component Analysis?PCA?.PCA of the metabolomics data suggested that the metabolites in 1000 mg/L C-dots treated plants were obviously distinct from untreated Arabidopsis.We examined the changes in Arabidopsis that occurred in the transcription of 33610genes represented in RNA-seq.After exposed with 1000 mg/L C-dots for 7 days,715 and 636up-regulated while 552 and 707 down-regulated genes?p-value<0.01,fold change?2?were identified in Arabidopsis roots and shoots,respectively.A significant overlap of differentially expressed genes was observed between Arabidopsis exposed with 125 and 1000 mg/L C-dots.Differentially expressed genes were classified in gene ontology?GO?categories using agriGO.Firstly,the up-regulated genes of 1000 mg/L C-dots exposure Arabidopsis were enriched in response to stimulus,secondary metabolic process,oxidoreductase activity,transmembrane transporter activity,transferase activity,endoplasmic reticulum lumen group et al.On the contrary,the GO analysis of down-regulated genes significantly enriched in photosynthesis,generation of precursor metabolites and energy,photosynthetic electron transport chain,chlorophyll binding and photosystem group et al.Mapman analysis of the differentially expressed genes suggested the similar response between Arabidopsis roots and shoots when exposed to C-dots.And C-dots could inhibite several metabolism pathways of photosynthesis.