Aluminium-fluoride Interactions In Tea Plants And The Mechanism Of Fluoride Toxicity In Arabidopsis Thaliana

Fluorine is an important halogen,which widely exists in the environment as fluoride compounds.Fluoride at high concentrations is toxic to plant cells,therefore inhibiting plant growth.However,tea(Camellia sinensis)plants grown in tea plantations are able to accumulate high levels of fluoride,especially in leaves,without showing F toxicity.The mechanisms of fluoride toxicity and tolerance in higher plants remain unclear.Tea is an acidophilic plant species growing on acidic soils that have high levels of available aluminium.Tea plants appear to require a certain amount of A1 for optimal growth and can accumulate large concentrations of Al.There is often a strong correlation between Al and F concentrations in tea plants.Al and F can form a number of soluble complexes AlFx3-x in solution.We hypothesize that the formation of AlFx3-x complexes affect F uptake and toxicity in tea plants.At present,little is known about the mechanism of fluoride toxicity and tolerance in higher plants,with previous research focusing on physiological observations.In order to answer the above questions,we conducted research in two parts:(1)To investigate the interactions between Al and F on growth,Al and F uptake and translocation,F speciation in tea plants.19F nuclear magnetic resonance(NMR)spectroscopy was used to determine F species in the nutrient solutions and the cell saps from root and leaf tissues.(2)A forward genetics approach was employed to screen F tolerant mutants in anethyl methane sulfonate(EMS)mutagenized population of Arabidopsis thaliana.This was followed by map-based cloning and high throughput sequencing to identify the mutated gene responsible for the fluoride tolerant phenotype.Detailed investigations,such as qRT-PCR analysis,tissue-specific expression were then carried out in order to elucidate the function of the gene.The main results are summarized as follows:(1)In hydroponic experiments,aluminium was found to stimulate roots growth of tea plants markedly,whereas F inhibited root and shoot growth.F caused necrosis of young leaf tips and the apex.The inhibitory effect of F was alleviated by the addition of Al to the nutrient solution.In a soil pot experiment,tea plants grew better when the Al to F molar ratio in leaves was higher than 1.When the ratio was lower than l,F toxicity symptoms occurred.The above results suggest that tea plants are sensitive to F toxicity and Al alleviates F toxicity.(2)In hydroponic experiments,A1 increased F concentration in roots,but decreased F concentration in leaves.The chemical forms of F in the nutrient solutions were determined by 19F-NMR spectroscopy.The results showed that the nutrient solutions containing only F treatment had a single strong peak with a chemical shift of 0 ppm,indicative of the free form of F-.In the solutions containing both F and Al,the 0 ppm peak disappeared and other peaks with chemical shifts of-37.2 ppm,-36.6 ppm and-35.5 ppm were detected,likely to represent AlF2+,AlF2+and AIF3,respectively,according to the speciation calculations using GEOCHEM.In the cell sap of roots and leaves exposed to only F treatment,a strong signal(0 ppm)was detected,representing free F-.Inclusion of Al in nutrient solution greatly suppressed the F signal at 0 ppm in both root and leaf sap.A small peak indicative of AlF2+was observed in the leaf sap.The above results suggest that Al alleviated F toxicity in tea by forming Al-F complexes,which affect the absorption and translocation of F.(3)Arabidopsis thaliana Col-0 seeds was exposed to EMS and M1 seeds was harvested.From the mutated seeds,four fluoride tolerant mutants(ftol1-4)were successfully obtained.200?M NaF was used in medium for its 50%inhibition to the primary root growth of Col-0.F1 test showed that the four mutants were allelic to each other.LPR2,encoding a multicopper oxidase(MCO),was cloned by map-based cloning and high throughput sequencing.A T-DNA insertional mutant of the LPR2 gene(lpr2)was obtained.Under 200?M NaF condition,lpr2 exhibited the same F-tolerant phenotype as that of ftol1.The wild-type LPR2 gene was transformed to ftol1,the transgenic plants showed the same F sensitive phenotype as the wild-type plants.The above results indicate that LPR2 is the casual gene for the F tolerant phenotype in ftol1 mutant.(4)LPR2 expression was detected by qRT-PCR and pLPR2:GUS.The results showed that LPR2 was expressed in shoots as well as roots.The expression of LPR2 was higher in shoots than roots.GUS staining results revealed that LPR2 was expressed strongly in leaf blade and the basal part of petiole.In the primary root,straining was detected in the places where lateral root primordia likely formed.The subcellular localization results revealed that LPR2 protein was present in the cytoplasm.The tissue and subcellular localization of LPR2 was different from those of LPR1,suggesting the functions of LPR1 and LPR2 are not redundant.(5)The phenotype of Col-0.Ipr1,ftol1 and lpr1 lpr2 double mutant exposed to different P,Fe and F treatments was compared.The results suggest that Fe availability in the medium is a critical factor for the low P response in the primary root growth of Arabidopsis.A sufficient supply of P is also important for the manifestation of the F-tolerant phenotype in ftol1,whereas the phenotype of LPR1 mutation was observed under low P conditions.It was also found that the phenotype of ftoll occurred only when Fe supply was abundant,and F exacerbated the Fe toxicity phenotype of the wild-type plants.When the medium contained abundant P and excess Fe,the change of Fe concentration and O2·-caused by additional F in Col-0 was not different from those in ftol1.However,additional F increased ferric iron content in the shoots of Col-0,but not in ftol1,suggesting that F aggravates Fe toxicity of Col-0 by affecting Fe oxidation state in plants.In summary,tea plants are sensitive to F toxicity in the absence of Al.A high availability of Al in acidic soils in tea plantations helps present F toxicity in tea plants.Aluminium alleviated F toxicity in tea by forming Al-F complexes and affecting the absorption and translocation of F.Under the conditions of abundant P and excess Fe,LPR2 is involved in Fe toxicity,probably by mediating the oxidation of Fe2+ to Fe3+,a process that is exacerbated by F stress.The findings from this study have advanced our understanding of F toxicity and tolerance in higher plants.