The μXRF maps indicate that Ce was mostly adsorbed on the barley roots or aggregated in the soil just outside the roots, but was not detected inside the roots . This data is similar with our previous finding which showed high concentration of Ce on wheat root surface.Hernandez et al. also reported CeO2-NPs adsorption in mesquite root grown in hydroponic culture. Selected hotspots were interrogated for Ce speciation, and showed that Ce was mostly in oxide form in spots analyzed in roots 1, 2 and 3 . LCF data showed that Ce was present mostly as CeO2 with small amounts of Ce species . The μXANES of CeO2 has a distinct shoulder on the low-energy side not found in other Ce species, and this shoulder is seen in all our spectra for which the signal is good enough to see it.We see no evidence for any Ce species other than CeO2. On the other hand, the μXANES of CeO2-NP differs very little from that of bulk CeO2, so we cannot distinguish bulk from nano and refer to both as CeO2. This data demonstrates that CeO2-NPs undergo very limited transformation in barley root-soil system, which is consistent with our findings in wheat rhizosphere that showed only 3–12% reduction to Ce species.Previous studies also showed similar results in transformation of CeO2-NPs to Ce acetate, carboxylate, phosphate, and hydroxide in plants .We found Ce signal near the tip of root 3, which indicates that a fraction of the CeO2 [Ce] were reduced to Ce. A second chemical map of root 3 extending further down from the root tip was acquired at longer x-ray dwell time of 200 milliseconds . Remarkably, root 3 spots 7, 8, 9,hydroponic nft system and 10 showed reduction of CeO2 to Ce species by 61, 77%, 56%, and 98%, respectively . μXRF map also revealed CeO2 inside barley root in the area close to where Ce was detected .

Root 3 spots 5 and 6 revealed that Ce was 89–91% CeO2 with small amount of Ce species . Based on this data, it is possible that Ce was taken up by barley roots as Ce, which got re-oxidized back to CeO2 inside the roots. Schwabe et al.reported that pumpkin and sunflower root exudates caused dissolution of CeO2-NPs that potentially facilitated the root uptake of Ce. Perhaps barley also produced extracellular compounds that reduced Ce and resulted in Ce uptake in roots. This is the first time that large reduction of CeO2-NPs to Ce has been recorded in soil. The highest value we found in literature was 48% Ce phosphate in soil, and 40% Ce phosphate and 34% Ce acetate in cucumber roots grown in hydroponic culture.As noted above, our previous study on wheat exposed to CeO2-NPs at similar soil and growing conditions used in the current experiment only showed 3–12% reduction of CeO2 to Ce species.10 Barley might have different root exudates than those from soybean, wheat, and cucumber in other experiments.Related study shows that plant type significantly alters concentrations and compositions of root exudates in stressed plants .The results also suggest that reduction sites for Ce may be highly localized since the huge amount of Ce was observed in root 3 only. This is consistent with reports showing that root exudate or oxidation-reduction sites tend to occur in isolated patches in roots.With regard to Ce → CeO2 [Ce] formation inside the roots, this finding is consistent with a report showing uptake of Ce ions that precipitated as CeO2-NPs in leaves.Current results are also in contrast with our findings in wheat wherein only CeO2-NPs, and no Ce, were detected in rhizosphere, and no Ce was found inside the roots.Both findings in wheat and barley corroborate our previous studies wherein above ground accumulation of Ce was recorded in barley but not in wheat.In summary, μXAS synchrotron spectroscopy revealed root surface adsorption and soil agglomeration of CeO2-NPs with transformation of Ce → Ce being highly localized in some roots.

The study revealed up to 98% reduction of CeO2-NPs to Ce species in soil-root interface which potentially facilitates root uptake of Ce species. These results provide additional insights into the mechanism of Ce transport and accumulation in plants. Owing to its pleasant flavor and multiple health benefits, tea is the second most popular nonalcoholic beverage worldwide, second only to water. Tea quality largely depends on the contents of polyphenols, caffeine, and the anine in the new shoots used for tea processing. Previous studies have shown that the biosynthesis of these metabolites is related to N conditions , with high quality tea being produced from plants grown under adequate N levels . However, high N fertilization accelerates soil acidification, which can lead to high accumulations of aluminum, fluorine, and heavy metals in tea leaves, posing potential risks to human health. In recent decades, tea produced from organic plantations has increased in popularity. For example, in China, during the past two decades, organic tea production has increased >45-fold . It has been shown that plants acquire N from the soil in the form of nitrate, ammonium, urea, and amino acids, with amino acids representing a significant N pool in some soils. Soil amino acids are derived mainly from exoenzymatic decomposition of proteins and peptides of decaying organisms. Tea plants constitute perennial crops whose leaves are harvested and are periodically pruned 2~3 times per year to maintain vigorous vegetative growth. In this regard, such tea plantation pruning has the potential to produce ~8000 kg ha−1 of pruned litterannually. This pruned litter contains high levels of amino acids and proteins that can be recycled following its decomposition in the soil. These pruned litter-derived amino acids may serve as an important N source to be taken up by tea plant root systems. Plant cells, including those of the roots, take up nutrients through a combination of passive and active transport mechanisms. Channels and permeases can participate in passive uptake when soil nutrient concentrations are high, whereas proton-coupled transporters engage in secondary active transport under low-nutrient conditions.

Given that soil amino-acid levels are much lower than those within the cells of roots, plasma membrane-localized transporters are generally required for amino-acid uptake from soils. At present, many plant amino-acid transporters have been identified and are grouped into two super families: amino acid/auxin permeases and amino acid-polyamine-choline transporters . The AAAP super family includes six families, amino-acid permeases , lysine and histidine transporters , proline transporters , GATs , auxin transporters , and aromatic and neutral amino-acid transporters ,nft channel whereas the APC super family includes members of the cationic amino-acid transporter and L-type aminoacid transporter families. Amino-acid transporters involved in uptake from the soil belong mainly to the AAP, ProT, and LHT families. In Arabidopsis, the LHT family members LHT1 and LHT6 have been shown to be critical for amino-acid uptake by plant roots. AtLHT1 displays uptake activity for glutamine , Ala, Glu, and Asp but not for Arg or Lys. AtLHT6 is involved in the uptake of acidic amino acids but does not seem to transport basic or other neutral amino acids. Studies devoted to amino-acid transporters have generally been performed on annual plant species, with much less information available on the molecular mechanisms underlying amino-acid transport in perennial species. In this study, we measured the amino-acid composition in the soil at a normal tea plantation and an organic tea plantation. Furthermore, we fed tea plants Glu, the most abundant amino acid detected in the soil and found that it was efficiently absorbed and utilized by these plants. We then cloned seven CsLHT genes and determined that CsLHT1 and CsLHT6 were able to transport a broad spectrum of amino acids. Arabidopsis lines over expressing CsLHT1 and CsLHT6 were found to exhibit increased uptake of exogenously supplied amino acids. In addition, expression patterns and subcellular localization studies provided support for the hypothesis that CsLHT1 and CsLHT6 play important roles in aminoacid uptake into the roots of tea plants.Tea plant seeds were grown in plastic pots filled with a mixture of soil and vermiculite . All the pots were irrigated weekly with tap water. The tea plant growth conditions included 16 h of light and 8 h of darkness, 70% relative humidity, and daytime and nighttime temperatures of 25°C and 18°C, respectively. After germination and growth for 100 d, healthy plants with similar crown sizes and heights were selected for studies performed under hydroponic culture. The hydroponic culture method was as described previously by Yang et al.. After 30 d in hydroponic culture, healthy plants with similar crown size and height were selected for amino-acid treatments. Nine seedlings were used for each treatment. For long-term feeding experiments, tea seedlings were transplanted into an N-deficient nutrient solution and allowed to grow for 2 d; afterward, a 1 mM Glu solution was added, and the seedlings were allowed to grow for 5 d. The addition of no Glu served as the control. The tea plant roots were then collected for amino-acid content analysis. For short-term feeding experiments, tea seedlings were transplanted into an Ndeficient nutrient solution and allowed to grow for 2 d; afterward, a 2 mM 15N-Glu solution was added, and the seedlings were allowed to grow for 6 h or 24 h. The addition of no 15N-Glu served as a control. The tea plant roots were then collected for gene expression analysis and 15N content determination using a DeltaV isotope ratio mass spectrometer . Amino-acid feeding to Arabidopsis was performed as previously described by Lee et al.. Arabidopsis seeds were rinsed in 70% ethanol for 1 min followed by sterilization in a solution containing 5% NaClO for 15 min.

The seeds were then washed four times using sterile water and then vernalized in water for 3 d at 4°C. For amino-acid uptake analysis, the seeds were cultured on 1/2-strength MS solid media for 1 week. The seedlings were then transferred to N-free 1/2-strength MS media that included either 1 mM 15N-Glu or 1 mM 15N-Gln and allowed to grow for 6 h. These Arabidopsis seedlings were then collected to determine the 15N content using a DeltaV isotope ratio mass spectrometer. The Arabidopsis growth conditions included 16 h of light and 8 h of darkness, 70% relative humidity, and daytime and nighttime temperatures of 21°C and 18°C, respectively.Amino-acid extraction was performed as previously described by Li et al.. Soil samples were collected from both a control fertilized tea plantation , located in Xuancheng, and an organic tea plantation , located in Hefei, Anhui Province, China.The normal tea plantation was fertilized, annually, whereas at the organic tea plantation chemical fertilizer had not been applied during the past 3 consecutive years. Soil samples were collected as follows: the humus in the surface soil was removed, and then soil to a depth of 10 cm was then collected, using a soil sampler; roots and other litter were then removed and 10 g soil samples were taken for drying, with three independent replicates being performed. Subsequently, 10 ml of water was added to each 4 g aliquot of dried soil and then incubated at 70°C for 12 h to extract amino acids. The samples were then cooled to room temperature, followed by centrifugation at 6000 × g for 10 min, after which the supernatants were then filtered through a 0.22 μm membrane. The filtrates were subsequently analyzed via an amino acid analyzer .Amino acids were extracted from 50 mg aliquots of freeze-dried tea plant roots using 5 ml of double-distilled water by boiling at 100°C for 20 min. The samples were then cooled to room temperature before centrifugation at 6000 × g for 10 min, after which the supernatants were then filtered through a 0.22 μm membrane. The filtrates were analyzed as described above.N is one of the most important mineral macro-nutrients essential for plant growth and development. In this regard, it is well known that plants have evolved the capacity to absorb N in the form of amino acid, from aqueous solutions. Furthermore, root uptake of amino acids is known to be energy dependent and regulated by the concentration of amino acids in solution, indicating that their uptake is an active process mediated by specific transporters. In this study, we determined that higher amino-acid levels are present in the soil of an organic tea plantation compared with a conventional tea plantation.