Contrasting Metabolisms in Green and White Leaf Sectors of Variegated Pelargonium zonale-An Integrative Transcriptomic and Metabolomic Study.

The photosynthetically active green leaf (GL) and non-active white leaf (WL) tissues of variegated Pelargonium zonale provide an excellent model system for studying processes associated with photosynthesis and sink-source interactions, enabling the same microenvironmental conditions. By combining differential transcriptomics and metabolomics, we identified the main differences between these two metabolically contrasting tissues. Genes related to photosynthesis and associated pigments, the Calvin-Benson cycle, fermentation, and glycolysis were strongly repressed in WL. On the other hand, genes related to nitrogen and protein metabolism, defence, cytoskeletal components (motor proteins), cell division, DNA replication, repair and recombination, chromatin remodelling, and histone modifications were upregulated in WL. A content of soluble sugars, TCA intermediates, ascorbate, and hydroxybenzoic acids was lower, while the concentration of free amino acids (AAs), hydroxycinnamic acids, and several quercetin and kaempferol glycosides was higher in WL than in GL. Therefore, WL presents a carbon sink and depends on photosynthetic and energy-generating processes in GL. Furthermore, the upregulated nitrogen metabolism in WL compensates for the insufficient energy from carbon metabolism by providing alternative respiratory substrates. At the same time, WL serves as nitrogen storage. Overall, our study provides a new genetic data resource for the use of this excellent model system and for ornamental pelargonium breeding and contributes to uncovering molecular mechanisms underlying variegation and its adaptive ecological value.

Desiccation Tolerance in Ramonda serbica Panc.: An Integrative Transcriptomic, Proteomic, Metabolite and Photosynthetic Study.

The resurrection plant Ramonda serbica Panc. survives long desiccation periods and fully recovers metabolic functions within one day upon watering. This study aimed to identify key candidates and pathways involved in desiccation tolerance in R. serbica. We combined differential transcriptomics and proteomics, phenolic and sugar analysis, FTIR analysis of the cell wall polymers, and detailed analysis of the photosynthetic electron transport (PET) chain. The proteomic analysis allowed the relative quantification of 1192 different protein groups, of which 408 were differentially abundant between hydrated (HL) and desiccated leaves (DL). Almost all differentially abundant proteins related to photosynthetic processes were less abundant, while chlorophyll fluorescence measurements implied shifting from linear PET to cyclic electron transport (CET). The levels of H2O2 scavenging enzymes, ascorbate-glutathione cycle components, catalases, peroxiredoxins, Fe-, and Mn superoxide dismutase (SOD) were reduced in DL. However, six germin-like proteins (GLPs), four Cu/ZnSOD isoforms, three polyphenol oxidases, and 22 late embryogenesis abundant proteins (LEAPs; mainly LEA4 and dehydrins), were desiccation-inducible. Desiccation provoked cell wall remodeling related to GLP-derived H2O2/HO● activity and pectin demethylesterification. This comprehensive study contributes to understanding the role and regulation of the main metabolic pathways during desiccation aiming at crop drought tolerance improvement.

In Silico Characterisation of the Late Embryogenesis Abundant (LEA) Protein Families and Their Role in Desiccation Tolerance in Ramonda serbica Panc.

Ramonda serbica Panc. is an ancient resurrection plant able to survive a long desiccation period and recover metabolic functions upon watering. The accumulation of protective late embryogenesis abundant proteins (LEAPs) is a desiccation tolerance hallmark. To propose their role in R. serbica desiccation tolerance, we structurally characterised LEAPs and evaluated LEA gene expression levels in hydrated and desiccated leaves. By integrating de novo transcriptomics and homologues LEAP domains, 318 R. serbica LEAPs were identified and classified according to their conserved motifs and phylogeny. The in silico analysis revealed that hydrophilic LEA4 proteins exhibited an exceptionally high tendency to form amphipathic α-helices. The most abundant, atypical LEA2 group contained more hydrophobic proteins predicted to fold into the defined globular domains. Within the desiccation-upregulated LEA genes, the majority encoded highly disordered DEH1, LEA1, LEA4.2, and LEA4.3 proteins, while the greatest portion of downregulated genes encoded LEA2.3 and LEA2.5 proteins. While dehydrins might chelate metals and bind DNA under water deficit, other intrinsically disordered LEAPs might participate in forming intracellular proteinaceous condensates or adopt amphipathic α-helical conformation, enabling them to stabilise desiccation-sensitive proteins and membranes. This comprehensive LEAPs structural characterisation is essential to understanding their function and regulation during desiccation aiming at crop drought tolerance improvement.

Alkaline soda Lake Velika Rusanda (Serbia): the first insight into diatom diversity of this extreme saline lake.

Alkaline soda lakes are unique habitats found in specific geographic regions, usually with dry climate. The Carpathian Basin is one of those regions very important for habitat and biodiversity conservation in Europe, with natural soda lakes found in Austria, Hungary and Serbia. In comparison to other two countries from Central Europe, algal biodiversity studies of saline soda lakes in Serbia are scarce. Lake Velika Rusanda has the highest measured salinity of all saline lakes in the Carpathian Basin and there were no reports of its diatom species richness and diversity till now. We conducted 2-year investigation programme to study biodiversity and seasonal dynamics of diatoms in this lake. A total of 27 diatom taxa were found, almost all of them attached to reed and much less in benthos and plankton. Five new diatom species for Serbia were recorded, Craticula halopannonica, Navicymbula pusilla, Hantzschia weyprechtii, Nitzschia thermaloides and Navicula staffordiae. The last mentioned is new for Europe as well. Lake Velika Rusanda is inhabited mostly by alkaliphilous and halophilic diatoms. Since diatoms are used as bioindicators in soda lakes, our results will improve their further application in ecological status assessment of these fragile habitats in the Carpathian Basin.

Size-segregated trace elements in continental suburban aerosols: seasonal variation and estimation of local, regional, and remote emission sources.

We have measured trace element contents in suburban aerosols from six size fractions in the range of PM0.27-16 from a background station in Belgrade (Serbia). The distribution and concentration of elements were determined within each of the investigated Dp fractions with emphasis on the fine and coarse modes. Fine/coarse mode ratios of element can provide information regarding their anthropogenic or natural origin. Analysis of seasonal variations of element contents in fine and coarse mode show that Cd, Co, K, and V have higher concentrations in the fine mode during heating season, while Fe contents are lower. In the coarse mode, Cu and V have higher concentrations during the heating season, while Al, Fe, Mg, Mn, and Sb contents are lower. We also apply a distribution probability model (normal, log-normal, and three-parameter Weibull) as new approach to estimate the distances of emission sources that can contribute to pollutant contents in particulate matter of the investigated location.

Partitioning of particulate matter and elements of suburban continental aerosols between fine and coarse modes.

The results presented in this work demonstrate for the first time a distribution of elements in the spectral analysis of aerosols in the suburban continental Balkan Peninsula. Samples were collected in the suburban area of Belgrade (Serbia) in the period from March 2012 till December 2013. Results presented here are from long-term measurements of masses of size-segregated aerosols and macro- and microelements in the range of PM0.27-16. The following elements were analyzed: Al, Ag, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, Pb, Sb, Ti, Tl, V, and Zn; levels of Be, Hg, and Se were under the detection limits in all samples. Average concentrations and time and seasonal variations of particulate matter (PM) as well as element contents and their percentage shares are given. The results showed the domination of particle content around the accumulation mode in the range of 0.53 < Dp < 1.06 µm, but the fractional distribution of elements showed maximal average concentrations in different fractions depending on the origin of each element. Crustal elements (Al, Ca, Fe, Mg, Mn, Ti, etc.) dominated in coarse mode, while anthropogenic elements (As, Cd, Cu, Pb, Sb, etc.) were mainly distributed in fine mode fractions. Some elements, such are As and Ni, were detected in investigated aerosols only occasionally, while others, such as Ca, Fe, and Mg, were detected in all analyzed samples. The application of multivariate analysis (PCA) demonstrated the connection between the elements of similar origin, in fine fractions mainly of anthropogenic origin, while in coarse mode of crustal origin, indicating the resuspension with contribution of about 40%. The contents of some measured elements were compared with their contents in aerosols in some European suburban areas.

Subacute alcohol and/or disulfiram intake affects bioelements and redox status in rat testes.

The aim of the study was to investigate if alcohol and disulfiram (DSF) individually and in combination affect bioelements' and red-ox homeostasis in testes of the exposed rats. The animals were divided into groups according to the duration of treatments (21 and/or 42 days): C21/C42 groups (controls); OL21 and OL22-42 groups (0.5 mL olive oil intake); A1-21 groups (3 mL 20% ethanol intake); DSF1-21 groups (178.5 mg DSF/kg/day intake); and A21+DSF22-42 groups (the DSF ingestion followed previous 21 days' treatment with alcohol). The measured parameters in testes included metals: zinc (Zn), copper (Cu), iron (Fe), magnesium (Mg) and selenium (Se); as well as oxidative stress (OS) parameters: superoxide anion radical (O2•-), glutathione reduced (GSH) and oxidized (GSSG), malondialdehyde (MDA), hydrogen peroxide (H2O2) decomposition and activities of total superoxide dismutase (tSOD), glutathione-S-transferase (GST) and glutathione reductase (GR). Metal status was changed in all experimental groups (Fe rose, Zn fell, while Cu increased in A21+DSF24-32 groups). Development of OS was demonstrated in A1-21 groups, but not in DSF1-21 groups. In A21+DSF22-42 groups, OS was partially reduced compared to A groups (A1-21>MDA>C; A1-21

Disulfiram moderately restores impaired hepatic redox status of rats subchronically exposed to cadmium.

Examination of cadmium (Cd) toxicity and disulfiram (DSF) effect on liver was focused on oxidative stress (OS), bioelements status, morphological and functional changes. Male Wistar rats were intraperitoneally treated with 1 mg CdCl2/kg BW/day; orally with 178.5 mg DSF/kg BW/day for 1, 3, 10 and 21 days; and co-exposed from 22nd to 42nd day. The co-exposure nearly restored previously suppressed total superoxide dismutase (SOD), catalase (CAT) and increased glutathione peroxidase (GPx) activities; increased previously reduced glutathione reductase (GR) and total glutathione-S-transferase (GST) activities; reduced previously increased superoxide anion radical (O2·-) and malondialdehyde (MDA) levels; increased zinc (Zn) and iron (Fe), and decreased copper (Cu) (yet above control value), while magnesium (Mg) was not affected; and decreased serum alanine aminotransferases (ALT) levels. Histopathological examination showed signs of inflammation process as previously demonstrated by exposure to Cd. Overall, we ascertained partial liver redox status improvement, compared with the formerly Cd-induced impact.

Oxidative stress, bioelements and androgen status in testes of rats subacutely exposed to cadmium.

The objective of our study was to examine testicular toxicity of cadmium (Cd), focusing on oxidative stress (OS), essential metals and androgenic status and morphological changes. Male Wistar rats [controls and four Cd-subgroups (n = 6) organized according to the exposure (1, 3, 10 and 21 days)] were intraperitoneally (i.p.) treated with 1 mg CdCl2/kg/day. Testicular Cd deposition was noticed from the 1st day. After 10 and 21 days, copper (Cu) and iron (Fe) increased by 60-109% and 43-67%, respectively, while zinc (Zn) decreased by 24-33%. During 1-21 days of the exposure, decrease in testicular total superoxide dismutase (SOD) and total glutathione-s-transferase (GST) activities occurred gradually by 30-78% and 15-84%, respectively, while superoxide anion radical (O2(-)) increased gradually by 114-271%. After 10-21 days, decrease in testicular catalase (CAT) activity appeared by 13-31%. After 21 days, malondialdehyde (MDA) decreased by 44% and the ratio of oxidized glutathione/reduced glutathione (GSSG/GSH) increased by 130% in testes of the rats exposed to Cd. Additionally, decreased testicular testosterone level and the relative testes mass, along with induced microscopic and macroscopic changes were occured, what can be explained as the consequence of instantly developed OS, impaired essential metals status and Cd testicular deposition.

Conventional, microwave, and ultrasound sequential extractions for the fractionation of metals in sediments within the Petrochemical Industry, Serbia.

In this paper, the main objective was fractionation of Cd, Cu, Ni, Pb, Zn, Ca, Fe, and K in certificate material and sediment samples gathered from and around the Petrochemical Industry using the conventional, microwave and ultrasonic sequential extraction. Microwave oven and ultrasound bath were used as an energy source for achieving faster extraction. Additional heating and boiling of samples were avoided by using lower power and shorter time for microwave and ultrasound extraction. Precision and accuracy of procedure were evaluated by using certificate material (BCR701). Acceptable accuracy of metals (87.0-111.3 %) was achieved for all three-step sequential of conventional extraction protocol. An accuracy of the fourth step has been verified with two certificate materials: BCR143R and 146R. The range of total extracted metal concentrations from sediments was similar for all three extraction techniques. A significant high percentage of Cd, Cu, and Zn were obtained after extraction of the exchangeable and acid soluble sediment fraction. Principal component analysis of values obtained after determination of risk assessment code using conventional and ultrasound sequential extraction show similarity of these values. Accuracy, recovery, and risk assessment code values imply that ultrasound sequential extraction is a more suitable, accelerated sequential extraction procedure (30 min per extraction step) than microwave extraction in applied conditions.