The aim of this study was to compare variety-dependent changes in the antioxidative status and fluorescence signatures of three basil varieties (Ocimum basilicum var. Genovese - GB, Ocimum & times; citriodorum - LB and Ocimum basilicum var. purpurascens - PB) grown under controlled light conditions in a growth chamber and in the open field. The main objective was to apply fluorescence spectroscopy combined with multivariate analysis to identify the key fluorophores present in various varieties, and their dynamics in changing environmental conditions. The combination of Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) method and principal component analysis (PCA) revealed distinct variety-specific responses to environmental growing conditions. Two key spectral components, C1 and C2, were resolved from fluorimetric measurements followed by multivariate analysis. While the shapes of the emission spectra of C1 remained consistent across all basil varieties and growing conditions, C2 exhibited variety-specific patterns, with lemon basil displaying a notable blue-shift towards shorter wavelengths under open field conditions, indicating a specific sensitivity to light stress. Metabolites known for their antioxidant activity (total phenolic and flavonoid content, ascorbate, and antioxidant capacity) were also quantified. Contrasted acclimatization strategies were demonstrated for the three varieties following sun exposure. In PB plants an increase in antioxidant activity was noticed in open field conditions. In contrast, an increase in ascorbate content was noticed in GB and LB varieties, along with increases in total phenolic content (TPC), total flavonoid content (TFC), and epidermal flavonoids (EpFlav) in GB, and without accompanying changes in antioxidant activity. Overall, fluorescence spectroscopy with MCR-ALS and PCA can be applied as a fast and non-invasive method for basil discrimination based on varieties and growing conditions depending on characteristic fluorophores. Obtained results indicate the potential of fluorescence-based approaches, combined with conventional antioxidant assays, as predictive markers of plant stress responses.

Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot, has a significant impact on cabbage production worldwide. The goal of this research was to evaluate the effect of preventive foliar treatments with Bacillus velezensis strain RD-FC 88 on the primary and secondary metabolism of Xcc-infected cabbage cv. Futo & scaron;ki plants. Special attention was given to measuring metabolites' changes, aiming to determine the influence of the applied biocontrol treatment on the development of plant immune response and resistance to pathogen. This study reports the first comprehensive biochemical and physiological analysis of the interaction between host plant, biocontrol strain and pathogen, thus providing novel insight into black rot management. Pathogen inoculation caused a significant decrease in the majority of measured metabolites, including most free amino acids (Gln, Ala, BCAA), phenolics, and glucosinolates. Preventive application of B. velezensis strain in Xcc-infected plants restored the levels of aromatic amino acids, Asp, Glu, Leu, Val, and Ala to control values. A similar pattern was observed in aliphatic glucosinolates sinigrin and glucoiberin, as well as for the indolic glucosinolate 4-methoxy-glucobrassicin. Additionally, increased accumulation of hydroxybenzoic acids, hydroxycinnamic acids, and kaempferol derivatives was also observed in the plants treated with the biocontrol strain and subsequently infected with Xcc, compared to plants solely infected with Xcc. The obtained results imply that the RD-FC 88 strain holds potential as an efficient priming agent, capable of stimulating cabbage cv. Futo & scaron;ki defense responses and enhancing its resistance to Xcc.

Quercus pubescens Willd. is a deciduous species that can retain leaves in the winter either as dead, standing leaves (marcescence), or as living, winter-green leaves. The retention of green leaves through winter is rare in continental areas. Winter-green leaves were observed on one-year-old seedlings of Q. pubescens in the winter of 2020 in the nursery of the Faculty of Forestry that lasted until the end of April 2021. The photosynthetic activity of photosystem II was measured using the rapid light curve method based on modulated pulse chlorophyll fluorescence. We assessed the potential photosynthetic activity of the leaves across the range of physiological stages: winter-green leaves retained from the previous growing season and on the leaves from the first, second, and third growth flush during the growing season. Photosystem II of winter-green leaves attained ~50-60% of the maximal photosynthetic activity obtained in spring and summer leaves, respectively. Climate data indicated that winter-green leaves exhibited frost resistance in the winter of 2020/2021, as their photosynthetic activity was preserved despite 33 days with sub-zero temperatures. The rapid light curve method also revealed the gradual acclimation of seedlings on a flush level, with leaves from the third flush best able to use available light under high temperatures and insolation efficiently. The results of rapid light use as an indicator of seedling acclimation are discussed. Some remarks on the possible practical significance of the winter-green leaves phenomenon (as in genetic selection) are highlighted.

Chloroplast movement rapidly ameliorates the effects of suboptimal light intensity by accumulating along the periclinal cell walls, as well as the effects of excess light by shifting to the anticlinal cell walls. These acclimation responses are triggered by phototropins located at the plasma membrane and chloroplast envelope. Here, we used a recently developed non-invasive system sensitive to very small changes in red light leaf transmittance to perform long-term continuous measurements of dark-light transitions. As a model system, we used variegated Pelargonium zonale leaves containing green sectors (GS) with fully developed chloroplasts and achlorophyllous, white sectors (WS) with undifferentiated plastids, and higher phototropin expression levels. We observed biphasic changes in the red-light transmittance and oscillations triggered by medium intensities of white light, described by a transient peak preceded by a constant decrease in transmittance level. A slight change in red-light transmittance was recorded even in WS. Furthermore, the chloroplast position at lower light intensities affected the rapid light curves, while high light intensity decreased saturated electron transport, maximum quantum efficiency of photosystem II, and increased non-photochemical quenching of chlorophyll fluorescence and epidermal flavonoids. Our results extend the knowledge of light-dependent chloroplast movements and thus contribute to a better understanding of their role in regulating photosynthesis under fluctuating light conditions.

Three basil plant varieties (Ocimum basilicum var. Genovese, Ocimum × citriodorum, and Ocimum basilicum var. purpurascens) were grown under moderate light (about 300 µmol photons m−2 s−1) in a glasshouse or growth chamber and then either transferred to an open field (average daily dose: 29.2 kJ m−2 d−1) or additionally exposed to UV-B irradiation in a growth chamber (29.16 kJ m−2 d−1), to reveal the variety-specific and light-specific acclimation responses. Total antioxidant capacity (TAC), phenolic profile, ascorbate content, and class III peroxidase (POD) activity were used to determine the antioxidant status of leaves under all four light regimes. Exposure to high solar irradiation at the open field resulted in an increase in TAC, total hydroxycinnamic acids (HCAs, especially caffeic acid), flavonoids, and epidermal UV-absorbing substances in all three varieties, as well as a two-fold increase in the leaf dry/fresh weight ratio. The supplemental UV-B irradiation induced preferential accumulation of HCAs (rosmarinic acid) over flavonoids, increased TAC and POD activity, but decreased the ascorbate content in the leaves, and inhibited the accumulation of epidermal flavonoids in all basil varieties. Furthermore, characteristic leaf curling and UV-B-induced inhibition of plant growth were observed in all basil varieties, while a pro-oxidant effect of UV-B was indicated with H2O2 accumulation in the leaves and spotty leaf browning. The extent of these morphological changes, and oxidative damage depended on the basil cultivar, implies a genotype-specific tolerance mechanism to high doses of UV-B irradiation.

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.

Since 2011, the outbreaks of brown rot caused by Ralstonia solanacearum race 3, biovar 2, phylotype IIB-1 (R3/B2/PIIB-1) have significantly compromised potato production in Serbia. During 6 years of monitoring (2013-2018) among 3,524 potato tuber samples, 344 were found positive for brown rot disease. R. solanacearum R3/B2/PIIB-1 was isolated from seven cultivars among 12 monitored, and in five localities among 17 monitored. Cultivar Lady Claire was found to have the highest disease frequency (31.98%). A total of 78 isolates were identified by R. solanacearum-specific primer pairs (PS-1/PS-2 and OLI-1/Y-2), as well as the following tests: restriction fragment length polymorphism analysis, biovar determination, immunofluorescence, biochemical analysis, and pathogenicity. The genetic composition of 36 selected isolates assessed using multilocus sequence analysis with seven genes (adk, gapA, gdhA, gyrB, ppsA, hrpB, and fliC) showed that all isolates originating from Serbian potato were homogeneous. By using the TCS algorithm of concatenated sequences to get insight into the phylogeography of isolates and other R. solanacearum strains deposited in the NCBI database, we showed that their origin is undetermined. Peroxidase (POD) activity was measured in brown rotted potato tubers. A positive correlation was found between POD activity and disease severity rated on the analysed tubers. In general, POD activity increased by 2-22 times in vascular necrotic tissues compared to non-necrotic ones, and depended on disease severity but not on cultivar. Native polyacrylamide gel electrophoresis analysis of POD profiles resulted in a total of 10 distinct POD isoforms, of which PODs 3-5 were highly intensified in response to R. solanacearum.

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.

The present study examined the effects of Candidatus Phytoplasma solani infection on antioxidative metabolism in leaves and roots of carrot (Daucus carota L.). Disease symptoms appeared at the end of June in the form of the chlorosis on some of the leaves, which became intensely red one week later, while the previously healthy leaves from the same branch becme chlorotic. A few days later, all leaves from the infected leaf branch were intensely red. Infected plants also had slower growth compared to the healthy ones with fewer leaf branches developed. The roots of infected plants were less developed, seared, or gummy with or without brown-colored root hair. The presence of the pathogen was detected by sequencing the 16S rRNA. National Center for Biotechnology Information (NCBI) BLAST analyses of the obtained sequence revealed 100% identity of tested strain with deposited Ca. Phytoplasma solani strains from various countries and hosts, all belonging to the "stolbur" group (16SrXII-A). Identity of 99.74% was found when the tested Serbian strain (MF503627) was compared with the reference stolbur strain STOL11 (AF248959). The oxidative damage of membranes in carrot cells was accompanied by a decrease in the content of photosynthetic pigments. Furthermore, for the determination of specific scavenging properties of the extracts, in vitro antioxidant assay was performed. In phytoplasma-infected carrot leaves, there was a greater reduction in the level of glutathione content (GSH); however; flavonoids and anthocyanidins seem to be responsible for the accompanied increased antioxidative capacity against hydroxyl radical and hydrogen peroxide.

Ascorbate oxidase (AO, EC 1.10.3.3) is a copper-containing enzyme localized at the apoplast, where it catalyzes the oxidation of ascorbic acid (AA) to dehydroascorbic acid (DHA) via monodehydroascorbic acid (MDHA) intermediate. Despite it has been extensively studied, no biological roles have been definitively ascribed. To understand the role of AO in plant metabolism, fruit growth and physiology, we suppressed AO expression in melon (Cucumis melo L.) fruit. Reduction of AO activity increased AA content in melon fruit, which is the result of repression of AA oxidation and simultaneous induction of certain biosynthetic and recycling genes. As a consequence, ascorbate redox state was altered in the apoplast. Interestingly, transgenic melon fruit displayed increased ethylene production rate coincided with elevated levels of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO, EC 1.14.17.4) activity and gene expression, which might contribute to earlier ripening. Moreover, AO suppressed transgenic melon fruit exhibited a dramatic arrest in fruit growth, due to a simultaneous decrease in fruit cell size and in plasmalemma (PM) ATPase activity. All the above, support for the first time, the in vivo AO participation in the rapid fruit growth of Cucurbitaceae and further suggest an alternative route for AA increase in ripening fruit.

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.

Resurrection plant Ramonda serbica is a suitable model to investigate vegetative desiccation tolerance. However, the detailed study of these mechanisms at the protein level is hampered by the severe tissue water loss, high amount of phenolics and polysaccharide, and possible protein modifications and aggregations during the extraction and purification steps. When applied to R. serbica leaves, widely used protein extraction protocols containing polyvinylpolypyrrolidone and ascorbate, as well as the phenol/SDS/buffer–based protocol recommended for recalcitrant plant tissues failed to eliminate persistent contamination and ensure high protein quality. Here we compared three protein extraction approaches aiming to establish the optimal one for both hydrated and desiccated R. serbica leaves. To evaluate the efficacy of these protocols by shotgun proteomics, we also created the first R. serbica annotated transcriptome database, available at http://www.biomed.unipd.it/filearrigoni/Trinity_Sample_RT2.fasta. The detergent-free phenol-based extraction combined with dodecyl-β-d-maltoside-assisted extraction enabled high-yield and high-purity protein extracts. The phenol-based protocol improved the protein-band resolution, band number, and intensity upon electrophoresis, and increased the protein yield and the number of identified peptides and protein groups by LC-MS/MS. Additionally, dodecyl-β-d-maltoside enabled solubilisation and identification of more membrane-associated proteins. The presented study paves the way for investigating the desiccation tolerance in R. serbica, and we recommend this protocol for similar recalcitrant plant material.

Bacterial leaf spot caused by the plant pathogenic bacterium Pseudomonas syringae pv. coriandricola (Psc) was observed on carrot, parsnip, and parsley grown on a vegetable farm in the Vojvodina Province of Serbia. Nonfluorescent bacterial colonies were isolated from diseased leaves and characterized using different molecular techniques. Repetitive element PCR fingerprinting with five oligonucleotide primers (BOX, ERIC, GTG(5), REP, and SERE) and the randomly amplified polymorphic DNA-PCR with the M13 primer revealed identical fingerprint patterns for all tested strains. Multilocus sequence analysis of four housekeeping genes (gapA, gltA, gyrB, and rpoD) showed a high degree (99.8 to 100%) of homology with sequences of Psc strains deposited in the Plant Associated Microbes Database and NCBI database. The tested strains caused bacterial leaf spot symptoms on all three host plants. Host-strain specificity was not found in cross-pathogenicity tests, but the plant response (peroxidase induction and chlorophyll bleaching) was more pronounced in carrot and parsley than in parsnip.

Trehalose and its precursor, trehalose 6-phosphate (T6P), are essential regulators of plant response to abiotic and biotic stress. Here we used the specific host-insect interaction between Linaria vulgaris (Plantaginaceae) and stem-galling weevil, Rhinusa pilosa (Mecinini, Curculionidae) with the aim to distinguish carbohydrate allocation patterns in response to herbivory, gall formation (G1, 24 h after oviposition), and gall development (G2, 7 days after oviposition) under controlled conditions. The hypothesis is that herbivory and galling induce distinct responses in both leaves and stems, and that shifts in carbon allocations are regulated by signaling sugars. Systemic response to herbivory was accumulation of T6P and maltose. The main feature of G1 in the stems was accumulation of trehalose, accompanied by increased T6P, turanose and glucose content, oppositely to the leaves. In G2, galls had 3-folds higher weight than controls, with further accumulation of fructose, glucose, turanose, and total water-insoluble carbohydrates (TIC), while the sucrose/hexose ratio decreased. Analysis of fast chlorophyll fluorescence kinetic (OJIP) transients in G2 showed a slight decrease in quantum yield of electron transport flux from Q(A) to Q(B), and towards photosystem I acceptor side, correlated with the decreased content of photosynthetic pigments and hexoses accumulation. Redistribution of photosynthates, and accumulation of T6P were induced in response to herbivory, indicating its signaling role. The results support the hypothesis that R. pilosa can induce plant reprogramming towards the accumulation of beneficial carbohydrates in developing gall by mechanisms which include both T6P and trehalose.

A new ground temperature profile sensor comprising of a row of thermistors enables a large number of equidistantly arranged measuring points. The row of thermistors was formed using a group of negative temperature coefficient thick film segmented thermistors connected in series. After that calibration of the DC thermistor resistance R was performed at room temperature and the sensor was placed in a hole drilled on the lawn. DC resistances R of the row of thermistors were measured versus depth z as a parameter and converted to actual temperature T(z) in the ground using the Steinhart-Hart equation. The temperature profile of the ground depending on deepness z and time t as a function T(t, z) was measured on different days during one year in the early morning and in the afternoon. The temperature at the ground surface was compared with the temperature of the deepest sensor point in the ground. Thermal diffusivity D of the ground was determined using measured data T(t, z) in the periods of steady ground temperatures. The new ground temperature profile sensor was realized to investigate the correlation between air and ground temperature, observation of ground temperature profile changes and heat exchange (heat transfer, diffusion, gradient, and heat flow direction) through the ground top layer and finally humidity estimation versus deepness.

We investigated metal accumulation in bamboo leaves during three seasons at three urban locations differing in pollution levels. The higher content of Cu, Pb, and Zn in the leaves was in correlation with the highest bioavailable content of these elements in the soil at the most polluted location. The content of leaf trace elements was higher in summer and autumn compared to spring. Scanning electron microscopy with energy dispersive X-ray spectroscopy showed that Si accumulation in bamboo leaves was the highest in epidermis and vascular tissue, and was co-localized with trace metals. Analysis of phytoliths showed co-deposition of Al, C, and Si, implying the involvement of Si in metal detoxification. Compared to a common urban tree, linden, bamboo showed better capacity to maintain cellular redox homeostasis under deteriorated environmental conditions. The results suggest that bamboo can be efficiently used for biomonitoring of air and soil metal pollution and remediation in urban areas.