Ksenija Radotić

Changes in peroxidase activity and isoenzymes in spruce needles after exposure to different concentrations of cadmium.

We studied the guaiacol peroxidase activity, isoenzyme pattern and metal content in the needles of 2-year-old spruce grown on soils supplemented with cadmium concentrations from 1 to 21 mg kg(-1). Following exposure to cadmium, an initial increase and subsequent decrease in the activity of the soluble fraction was observed. A parallel change of their isoenzyme pattern occurred. An increase of the cell wall-bound peroxidase activity under prolonged metal treatment was evident. The results obtained show that peroxidase activity and isoenzyme pattern could be used to evaluate the capacity of one part of the defense system in spruce seedlings to withstand metal stress.

Atomic Force Microscopy Stiffness Tomography on Living Arabidopsis thaliana Cells Reveals the Mechanical Properties of Surface and Deep Cell-Wall Layers during Growth

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content.

Quantification of compression wood severity in tracheids of Pinus radiata D. Don using confocal fluorescence imaging and spectral deconvolution.

Confocal fluorescence microscopy was used to examine the spectral characteristics of lignin autofluorescence in secondary cell walls of normal and compression wood from Pinus radiata. Using UV excitation, fluorescence spectra of normal and compression wood sections showed significant differences, especially in the outer secondary cell wall of tracheids, with a shift in maxima from violet to blue wavelengths between normal and compression wood. A comparison of normal wood, mild and severe compression wood, showed that the wavelength shift was intermediate in the mild compression wood compared to the severe compression wood, thus offering the possibility of quantifying the severity by measuring ratios of fluorescence at violet and blue wavelengths. Fluorescence induced by blue light, rather than UV, was less well differentiated amongst wood types. Spectral deconvolution indicated the presence of a minimum of five discrete lignin fluorophores in the cell walls of both normal and compression wood tracheids. Comparison with lignin model compounds suggest that the wavelength shift may correspond in part to increased levels of p-hydroxy type lignin in the compression wood samples. The combination of confocal fluorescence imaging and related spectral deconvolution therefore offers a novel technique for characterising cell wall lignin in situ.

Luminescent carbon nanoparticles: effects of chemical functionalization, and evaluation of Ag+ sensing properties

Luminescent carbon-based nanoparticles, addressed as carbon dots (CDs), were synthesized at relatively low temperature from lactose following an easy and inexpensive procedure. Modification of their surface was done by functionalization with mercaptosuccinic acid (MSA) (CDs–MSA). Transmission electron microscopy images showed regular spherical nanoparticles of 5 nm diameter. Raw and functionalized (CDs–MSA) CDs were highly fluorescent at 448 and 472 nm, with relative high quantum yield (Φ = 0.21 and 0.46 respectively). At the maximum fluorescence of CDs–MSA the intensity was quenched by addition of Ag+ ions by a static mechanism with a Stern–Volmer constant of 3.7 × 103 M−1. The analysis of the emission spectra showed that the CD–MSA complex was stable after this process. The quenching profiles showed that only 44% of the CD–MSA fluorophores are accessible to Ag+. The main figures of merit were detection and quantification limits of 385.8 nM and 1.2 μM respectively, and the precision as relative standard deviation was 1.76%. No interference was observed when other metal ions were present indicating a high selectivity for Ag+ detection. The results showed that CDs–MSA can be used for efficient quantification of Ag+ in silver nanoparticle dissolution.

Fluorescence lifetime imaging of lignin autofluorescence in normal and compression wood

Wood cell walls fluoresce as a result of UV and visible light excitation due to the presence of lignin. Fluorescence spectroscopy has revealed characteristic spectral differences in various wood types, notably normal and compression wood. In order to extend this method of characterising cell walls we examined the fluorescence lifetime of wood cell walls using TCSPC (Time‐Correlated Single Photon Counting) as a method of potentially detecting differences in lignin composition and measuring the molecular environment within cell walls. The fluorescence decay curves of both normal and compression wood from pine contain three exponential decay components with a mean lifetime of τm = 473 ps in normal wood and 418 ps in compression wood. Lifetimes are spatially resolved to different cell wall layers or cell types where individual lifetimes are shown to have a log‐normal distribution. The differences in fluorescence lifetime observed in pine compression wood compared to normal wood, are associated with known differences in cell wall composition such as increased p‐hydroxyphenyl content in lignin as well as novel deposition of β(1,4)‐Galactan. Our results indicate increased deposition of lignin fluorophores with shorter lifetimes in the outer secondary wall of compression wood. We have demonstrated the usefulness of fluorescence lifetime imaging for characterising wood cell walls, offering some advantages over conventional fluorescence imaging/spectroscopy. For example, we have measured significant changes in fluorescence lifetime resulting from changes to lignin composition as a result of compression wood formation that complement similar changes in fluorescence intensity.

Probing the lignin nanomechanical properties and lignin–lignin interactions using the atomic force microscopy

By combining atomic force microscopy (AFM) force and environmental scanning electron microscopies (ESEMs), herein we present an evidence for the existence of strong intermolecular forces, which are responsible for holding lignin globules together in higher ordered structures. Based on this observation, we provide a support for the hypothesis that lignin globules consist of at least two individual spherical layers, with space in between filled with solvent or gas.

A study of lignin formation at the molecular level by scanning tunneling microscopy

A scanning tunneling microscope (STM) was used to observe the temporal formation and organization of dehydrogenative polymer (DHP) synthesized from coniferyl alcohol. The images obtained elucidate this structure for the first time. The structure of DHP, as seen from STM images, shows long-range order. It appears that DHP consists of building units or modules assembled into larger assemblies called supermodules. Supermodules are interconnected into the overall lattice-like polymer structure with or without spherical regions. One module consists of about 20 monomers, while the supermodule contains about 500 monomers. Calculated molecular weights for modules and supermodules agree with DHP molecular weight distribution peaks. Samples prepared at two different pH values, 6.4 and 7.6, have the same characteristics. The results presented demonstrate that the process of lignification, even in in vitro conditions, is highly ordered, and as such contribute to our understanding of the structure of lignin, a significant constitutive and functional element of cell walls.

Activities of Antioxidant Systems During Germination of Chenopodium rubrum Seeds

The activities of superoxide-dismutase (SOD), catalase (CAT) and peroxidase (POD), and concentrations of glutathione and ascorbate have been studied during the first stages of germination in Chenopodium rubrum L. seeds. The highest CAT and SOD activity was found prior to radicle protrusion, while POD activity was maximal at the time of radicle protrusion and seedling development, new POD isozymes simultaneously appearing. The concentrations of total, reduced and oxidized glutathione showed similar changes during germination, the highest values being detected at the time of radicle protrusion. Ascorbic acid was present in the seeds in a detectable concentration only at the time preceding radicle protrusion, while its oxidized form dehydroascorbic acid was detected during the whole germination period studied. Gibberellic acid (GA3, 160 μM) had no effect on germination percentage, but in presence of GA3, SOD and CAT activity notably increased prior to radicle protrusion, and oxidized glutathione concentration decreased in further germination.

Anti-cancer effects of cerium oxide nanoparticles and its intracellular redox activity

Data on medical applications of cerium oxide nanoparticles CeO2 (CONP) are promising, yet information regarding their action in cells is incomplete and there are conflicting reports about in vitro toxicity. Herein, we have studied cytotoxic effect of CONP in several cancer and normal cell lines and their potential to change intracellular redox status. The IC50 was achieved only in two of eight tested cell lines, melanoma 518A2 and colorectal adenocarcinoma HT-29. Self-propagating room temperature method was applied to produce CONP with an average crystalline size of 4 nm. The results confirmed presence of Ce(3+) and O(2-) vacancies. The induction of cell death by CONP and the production of reactive oxygen species (ROS) were analyzed by flow-cytometry. Free radicals related antioxidant capacity of the cells was studied by the reduction of stable free radical TEMPONE using electron spin resonance spectroscopy. CONP showed low or moderate cytotoxicity in cancer cell lines: adenocarcinoma DLD1 and multi-drug resistant DLD1-TxR, non-small cell lung carcinoma NCI-H460 and multi-drug resistant NCI-H460/R, while normal cell lines (keratinocytes HaCaT, lung fetal fibroblasts MRC-5) were insensitive. The most sensitive were 518A2 melanoma and HT-29 colorectal adenocarcinoma cell lines, with the IC50 values being between 100 and 200 μM. Decreased rate of TEMPONE reduction and increased production of certain ROS species (peroxynitrite and hydrogen peroxide anion) indicates that free radical metabolism, thus redox status was changed, and antioxidant capacity damaged in the CONP treated 518A2 and HT-29 cells. In conclusion, changes in intracellular redox status induced by CONP are partly attributed to the prooxidant activity of the nanoparticles. Further, ROS induced cell damages might eventually lead to the cell death. However, low inhibitory potential of CONP in the other human cell lines tested indicates that CONP may be safe for human usage in industry and medicine.

Further in vitro evaluation of antiradical and antimicrobial activities of phytol

The antiradical activity of phytol was evaluated by electron paramagnetic resonance towards hydroxyl radical (√OH), superoxide anion radical (), methoxy radical (√CH2OH), carbon-dioxide anion radical (), as well as towards nitric-oxide radical (√NO) and 2,2-diphenyl-1-picrylhydrazyl (√DPPH) radical. It reduced the production of all tested radicals showing more promising activity against , √CH2OH and √DPPH radicals (56%, 50% and 48%, respectively) in comparison with √NO, and √OH radicals (38%, 23% and 15%, respectively). The antimicrobial activity of phytol was evaluated by the microdilution method against eight bacterial and eight fungal strains. To varying degrees, it was proven to be active against all tested bacteria and fungi (MIC 0.003–0.038 mg/mL and MBC 0.013–0.052 mg/mL, MIC 0.008–0.016 mg/mL and MFC 0.090–0.520 mg/mL, respectively). According to the obtained results, medical foods containing phytol may support development of new therapies for heart disease.