Search

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.

Potato blackleg is frequently observed on the production fields in the Backa region of Vojvodina province, which is one of the largest potato-growing areas in Serbia. This disease usually occurs during June and July. In July 2020, blackleg symptoms in the form of stem necrotic lesions, vascular discoloration, hollow stems, and wilting of whole plants were noted on potato cultivar VR808 on a field 28 ha in size located in Maglic village (GPS coordinates 45.349325 N, 19.542768 E). Disease incidence was estimated at 20-25%. Isolations were performed from 12 potato samples on Crystal Violet Pectate medium (CVP). Stem sections consisted of brown lesions and healthy tissue (c.10 cm) were surface sterilized with ethyl alcohol 70% (w/v) and rinsed with sterile distilled water. Small pieces of tissue were taken at the edges of stem lesions (between healthy and diseased tissue) were soaked in phosphate buffer saline for 20 min and plated using a standard procedure (Klement et al. 1990). Single colonies that formed pits after 48 hours at 26 °C were re-streaked onto Nutrient Agar (NA) where creamy white colonies with smooth surfaces were formed. A total of 30 isolates were selected and DNA isolated from the colonies was further analyzed by polymerase chain reaction (PCR) using the partial dnaX gene (DNA polymerase subunit III gamma/tau) with primer pair dnaXf/dnaXr for Pectobacterium and Dickeya species identification (Slawiak et al. 2009). A single characteristic band of 535 bp was amplified in all isolates (Slawiak et al. 2009). DNA sequence alignment showed two distinct groups of isolates (Fig.S1), which were genetically uniform within each group. Using BLASTn search, it was established that the dnaX sequence of the first group (consisting of 19 Serbian potato isolates) had 99.79% identity with NCBI-deposited Pectobacterium versatile strains 14A and 3-2 from potato from Belarus (Acc. No. CP034276 and CP024842, respectively) as well as SCC1 from Finland (Acc. No. CP021894). The remaining 11 dnaX sequences had 100% identity with Pectobacterium carotovorum subsp. carotovorum strain CFBP7081 originating from water in Spain (Acc. No. MK516961). The partial dnaX sequences of three Serbian P. versatile isolates (Pv1320, Pv1520, and Pv1620) and one P. carotovorum subsp. carotovorum (Pcc2520) were deposited in GenBank under Acc. No. MW839571, MW805306, MW839572, and MW805307, respectively. These results, indicating combined infection in the observed field, signify the first identification of P. versatile in Serbia. Multilocus sequence analysis (MLSA) performed with proA (proAF1/ proAR1) and mdh (mdh2/mdh4) genes (Ma et al. 2007; Moleleki et al. 2013) grouped three tested Serbian potato P. versatile isolates together with P. versatile strains from NCBI (Fig.S2). For both tested genes, BLASTn search revealed 100% homology with P. versatile strain SCC1 from Finland. Three Serbian P. versatile potato isolates were deposited under Acc. Nos. MZ682623-25 for proA and MZ682620-22 for mdh genes. According to the routine tests suggested for Pectobacteriaceae (Schaad et al. 2001), Serbian isolates possessed microbiological traits identical to P. versatile description (Portier et al. 2019). Pathogenicity was performed on potato cultivar VR808 with three selected P. versatile isolates (Pv1320, Pv1520, and Pv1620) in the following assays: (i) surface-sterilized tuber slices with holes in the center filled with 100 µL of bacterial suspensions (adjusted to 109 CFU mL-1) to test the isolates' ability to cause soft rot, and (ii) young, four-week old plants with developed 3rd true leaf (c. 30 cm tall) were inoculated by injecting stems with bacterial suspension adjusted to 107 - 108 CFU mL-1 at a height 5 cm above the soil line. Negative controls were treated with sterile distilled water. Inoculated plants were kept under controlled conditions (25 °C temperature and >70% relative humidity). Each assay was replicated twice. Soft rot appeared on tuber slices 24 h after inoculation. On inoculated stems, initial symptoms manifested as greasy elongated spots at inoculation sites two days after inoculation (DAI), and subsequently extended along the vascular tissue and became necrotic. Whole plant's decay was recorded in five DAI, while negative controls remained healthy. To complete Koch's postulates, bacteria were re-isolated from symptomatic potato plants and confirmed by PCR and sequencing of dnaX. This first report of P. versatile in potato indicates that blackleg currently present in Serbia is caused by a diverse bacterial population. This pathogen was first identified in genome comparison as 'Candidatus Pectobacterium maceratum' (Shirshikov et al. 2018) and was later renamed as Pectobacterium versatile sp. nov. (Portier et al. 2019). Thus far, bacterium Pectobacterium carotovorum subsp. brasiliensis has been recognized as dominant pathogen on most of the infected fields in Vojvodina province, and was recently noted on one plot subjected to a combined infection with Dickeya dianthicola (Markovic et al. 2021). Findings achieved in this study are highly relevant, as they point to the diversity in potato blackleg pathogens, likely due to the increasingly widespread distribution of imported seed potatoes.

Ramonda serbica Panc. is a resurrection plant that can survive long desiccation periods (extreme loss of cellular water). The accumulation of late embryogenesis abundant proteins (LEAPs) is a crucial step in desiccation tolerance mechanism. Based on in vitro studies, LEAPs can be involved in antioxidative defense, ion sequestration, structural stabilization of both membranes and enzymes during freezing or drying, while by forming intracellular proteinaceous condensates they increase structural integrity and intracellular viscosity of cells during desiccation. Here we investigated the antioxidative potential of LEAPs identified by de novo transcriptomics of R. serbica, based on their primary and secondary confirmation. In our previous work [1], we displayed the antioxidative capacity of 20 free proteogenic amino acids (FAA) through determining their hydroxyl radical (•OH, generated in Fenton reaction) scavenging rate by using electron paramagnetic resonance. These results served as a basis for generating a model for prediction of •OH scavenging activity for selected proteins. In addition, the model was built based on protein primary sequences, hydrophobicity, 3D structure and predicted solvent accessible area. Manually curated data for peptides and proteins with experimentally determined •OH scavenging rate were used for training and testing. The model was fed into machine learning algorithm and •OH scavenging potential scale was created using IC50 values. By applying our model, we classified 164 LEAPs according to their potential for •OH scavenging. Further work will focus on the experimental evaluation of the obtained model by measuring of the rate of • OH scavenging in the presence of recombinantly produced LEAPs. Organized by Society for Free Radical Research Europe (SFRR-E).

Two tomato genotypes with constitutively different ABA level, flacca mutant and wild type of Ailsa Craig cv. (WT), were subjected to three repeated drought cycles, with the aim to reveal the role of the abscisic acid (ABA) threshold in developing drought tolerance. Differential responses to drought of two genotypes were obtained: more pronounced stomatal closure, ABA biosynthesis and proline accumulation in WT compared to the mutant were compensated by dry weight accumulation accompanied by transient redox disbalance in flacca. Fourier-transform infrared (FTIR) spectra analysis of isolated cell wall material and morphological parameter measurements on tomato leaves indicated changes in dry weight accumulation and carbon re-allocation to cell wall constituents in flacca, but not in WT. A higher proportion of cellulose, pectin and lignin in isolated cell walls from flacca leaves further increased with repeated drought cycles. Different ABA-dependent stomatal closure between drought cycles implies that acquisition of stomatal sensitivity may be a part of stress memory mechanism developed under given conditions. The regulatory role of ABA in the cell wall restructuring and growth regulation under low leaf potential was discussed with emphasis on the beneficial effects of drought priming in developing differential defense strategies against drought.

Water deficit has a global impact on plant growth and crop yield. Climate changes are going to increase the intensity, duration and frequency of severe droughts, particularly in southern and south-eastern Europe, elevating the water scarcity issues. We aimed to assess the contribution of endogenous abscisic acid (ABA) in the protective mechanisms against water deficit, including stomatal conductance, relative water potential and the accumulation of osmoprotectants, as well as on growth parameters. To achieve that, we used a suitable model system, ABA-deficient tomato mutant, flacca and its parental line. Flacca mutant exhibited constitutively higher levels of soluble sugars (e.g., galactose, arabinose, sorbitol) and free amino acids (AAs) compared with the wild type (WT). Water deficit provoked the strong accumulation of proline in both genotypes, and total soluble sugars only in flacca. Upon re-watering, these osmolytes returned to the initial levels in both genotypes. Our results indicate that flacca compensated higher stomatal conductance with a higher constitutive level of free sugars and AAs. Additionally, we suggest that the accumulation of AAs, particularly proline and its precursors and specific branched-chain AAs in both, glucose and sucrose in flacca, and sorbitol in WT, could contribute to maintaining growth rate during water deficit and recovery in both tomato genotypes.

Tomato (Solanum lycopersicum L.) is among the economically most important vegetables in Europe, valued for its bioactive properties due to significant contents of vitamins, carotenoids, and phenolic compounds. In this study, the tomato cultivar Big Beef F1 was grown in the open field (OF) and under polytunnels in central Serbia during 3 years. Polytunnels were covered with two foils (both with 57% reduced photosynthetic active radiation, PAR) differing in UV-A and UV-B transmittance. The aim of our work was to determine the influence of light conditions on accumulation of phytonutrients (carotenoids and phenolics) in the peel and flesh of ripe tomato fruits. The amount of effective antioxidants, caffeic acid, and quercetin (phenolics with ortho-dihydroxy substitution) in the peel was the highest in tomato fruits grown in the OF (maximal PAR and UV-A and UV-B radiation). Moreover, the content of leaf epidermal flavonoids was the highest in the OF. The content of lycopene and β-carotene in the flesh of tomato fruit was higher under the polytunnel with higher UV-transmittance. Our results showed that selection of the right light conditions (quality and intensity) for tomato production has a significant effect on the accumulation of beneficial phenolics and carotenoids.

In this study we exposed variegated leaves of Pelargonium zonale to strong sunlight (>1100 μmol m −2 s −1 of photosynthetically active radiation) with and without paraquat (Pq), with the aim to elucidate the mechanisms of H 2 O 2 regulation in green and white tissues with respect to the photosynthetically-dependent generation of reactive oxygen species (ROS). Sunlight induced marked accumulation of H 2 O 2 in the apoplast of vascular and (peri)vascular tissues only in green sectors. This effect was enhanced by the addition of Pq. In the presence of diphenyl iodide, an NADPH oxidase inhibitor, H 2 O 2 accumulation was abolished. Distinct light-induced responses were observed: in photosynthetic cells, sunlight rapidly provoked ascorbate (Asc) biosynthesis and an increase of glutathione reductase (GR) and catalase activities, while in non-photosynthetic cells, early up-regulation of soluble ascorbate peroxidase, dehydroascorbate reductase (DHAR) and GR activities was observed. Paraquat addition stimulated DHAR and GR activities in green sectors, while in white sectors activities of monodehydroascorbate reductase, DHAR and class III peroxidases, as well as Asc content rapidly increased. Differential antioxidative responses in the two tissues in the frame of their contrasting metabolisms, and the possible role of (peri)vascular H 2 O 2 in signaling were discussed.

Iron–organic interactions are involved in a variety of environmental phenomena, including photo-redox reactions, iron cycling and bioavailability, as well as contaminant fate. In this study we examined UV-induced redox reactions of iron and indole in water. The presence of one indole in the irradiated system resulted in the presence of eight reduced ferric ions, not counting direct photolysis of Fe3+ complexes with OH−, which gives Fe2+ and hydroxyl radical (HO ) as products. The main mechanisms that contribute to indole-related Fe3+ reduction i.e. Fe2+ accumulation are: (i) HO scavenging, which prevents oxidation of Fe2+ by HO ; (ii) oxidation of indole and its derivatives by excited ferric iron; (iii) reduction of ferric iron by excited indole (not present under UV-A). Hydrated electrons released by UV-B-excited indole play only a minor role in the reduction of iron. Indole-derived radicals emerged as byproducts of indole/iron photo-chemistry. 1H NMR and low-T EPR spectroscopy showed that indole forms a weak low-symmetry complex with Fe3+. The strongest interactions between iron and π-cloud in the indole ring are at positions 2, 3, and 7. The formation of complex promotes electron transfer from excited indole to Fe3+. Our findings are important for understanding the catalysis of photo-reduction of iron by heterocyclic aromatic pollutants, and for the development of protocols for indole processing in wastewaters.

In this study we exposed variegated leaves of Pelargonium zonale to strong sunlight (1100μmolm