Leaf senescence constitutes a highly regulated final phase of leaf development, leading to cell death that is recognized as a type of programmed cell death (PCD). Degradation of nuclear DNA into ...oligonucleosomal fragments (DNA ladder) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay are methods commonly used to detect PCD-specific DNA cleavage. TUNEL reaction in situ labels free 3'-OH DNA strand breaks (nicks), thus allowing histological localization of nuclear DNA degradation during PCD. Here we describe in situ labeling of PCD-specific nuclear DNA fragmentation on conventional histological sections of senescing tobacco leaves. Incorporation of fluorescein-labeled dUTPs is detected by fluorescence microscopy, which enables in situ visualization of PCD at the single-cell level in the leaf mesophyll tissues undergoing senescence.
Pinus peuce (Macedonian pine) is considered a valuable ornamental tree that is frequently planted in parks and gardens, especially in Western Europe. This endemic pine is one of the most valuable ...conifer species in its native range, which currently consists of only two disjunct populations restricted to small mountainous areas of the Balkans and is listed as a near-threatened species. The reproduction of Macedonian pine by seed is limited, so in vitro propagation methods have emerged as a promising tool for large-scale propagation. The objective of this study was to develop an improved system for the micropropagation of P. peuce from juvenile plant material using a short-term liquid cytokinin pulse. For that, explants derived from 4-week-old seedlings were pulse-treated with different concentrations of N6-benzyladenine (BA) for 1 or 2 h to stimulate the induction of axillary buds. The highest axillary shoot formation was achieved with 222 µM BA pulse treatment, with an average number of ~six shoots per explant. Elongated shoots (≥10 mm) were detached from the explants and pulse-treated with 0.27 or 1.08 mM α-naphthaleneacetic acid (NAA) or 0.25 or 0.98 mM indole-3-butyric acid (IBA) for 1 or 2 h. IBA was more effective than NAA and led to a maximum rooting percentage (up to 40%) and the highest number of acclimatized plants (15–20%). Rooted plants were successfully transferred to ex vitro conditions.
(Rochel) Bräuchler (Lamiaceae) is an endangered species endemic to the Southern Carpathians. It is characterized by the production of high amounts of essential oils, which emit volatile organic ...compounds (VOCs) that have an essential role in biotic and abiotic stress responses and in plant-plant and plant-insect interactions. The present study was initiated to phytochemically examine the influence of different carbon sources in the nutrition medium on VOC emissions of micropropagated
plants, using gas chromatography-mass spectrometry analysis of headspace VOCs. The volatile profiles were subjected to multivariate analysis with respect to the presence, concentration and type of carbon source in the nutrient medium. In addition, the effect of different carbohydrates on the density and size of the leaf glandular trichomes, the main structures involved in the emission of VOCs, was determined. A total of 19 VOCs, primarily belonging to mono- and sesquiterpenes previously described in plants, were tentatively identified. Six VOCs were produced at levels higher than 2% of the total VOC emission, dominated by pulegone, ß-pinene and menthone. Inclusion of the carbohydrates in the culture media affected the production of the main leaf trichome-associated volatile allelochemicals although the qualitative composition of the volatiles changed only slightly. Multivariate analysis showed that the concentration, rather than the carbohydrate type, influenced the VOC profile.
Micromeria spp. are aromatic plants, many of which were shown to exhibit various biological effects. The present study aimed to determine the content and the composition of the essential oil of in ...vitro-cultured Micromeria croatica (Pers.) Schott and to evaluate the in vitro antimicrobial activity of its methanolic extract, in order to compare its phytochemical profile and biological activity with wild-growing plants. Shoots regenerated on MS medium without plant growth regulators (PGRs) or supplemented with kinetin were used for phytochemical analysis. Essential oils from both native plant material and in vitro-cultivated M. croatica plants, with a total of 44 identified constituents, were dominated by oxygenated monoterpenes. Borneol was the main component detected in wild-growing plants (25.28%) and micropropagated plants grown on PGR-free medium (20.30%). Kinetin treatment led to increased oil yield and favored the production of oxygenated monoterpenes, dominated by geranial (33.53%) and cis-p-mentha-1 (7), 8-dien-ol (23.69%). The percentage of total sesquiterpenoids in micropropagated plant material was considerably lower than in wild-growing plants. In vitro culture conditions and PGRs affected the production of essential oils, inducing quantitative modifications in the production of the major volatile constituents in M. croatica plantlets. The antimicrobial activity of M. croatica methanolic extracts was investigated using the broth microdilution method. Extracts obtained from in vitro cultures generally exhibited greater antibacterial potential, compared to wild-growing plants. Among six bacterial strains tested, Bacillus cereus and Staphylococcus aureus were the most sensitive microorganisms. The present study provided evidence that in vitro culture conditions might favorably affect the antimicrobial activity of M. croatica methanolic extracts.
The anatomy and ultrastructure of the short glandular trichomes occurring on young expanding leaves of Nicotiana tabacum were investigated using light and transmission electron microscopy. The ...objective of the present research was to characterize the cellular changes that occur during morphogenesis of short glandular trichomes, from initiation to senescence. Ultrastructural analysis of their secretory cells revealed characteristics common to gland cells: numerous mitochondria in highly organized cytoplasm, large nuclei, and an elaborate network of endoplasmic reticulum. Initial changes in nuclear and plastidial organization were observed at a more advanced secretory stage, marking the onset of senescence. During trichome senescence, gradual reduction of the cytoplasm density occurred along with structural changes of the plastids and the tonoplast. As a result of inward blebbing of the cytoplasm into the vacuole, membrane bound vesicular structures appeared in the vacuolar space. At the late secretory stage, marked by an increase in vacuolation and extraplasmic space, degenerative changes included further fragmentation of the cytoplasm and deterioration of the tonoplast. Multimembrane myelin bodies observed in the vacuolar space were indicative of membrane digestion although plasma membrane did not appear massively degraded.
Cistus creticus subsp. creticus is a shrubby Mediterranean plant used since ancient times in folk medicine for the treatment of different diseases. C. creticus extracts and resin contain different ...types of secondary metabolites, such as terpenoids (predominantly labdane type diterpenoids), and phenylpropanoids. Growth conditions seem to influence the content of labdane-type diterpenes and flavan-3-ols in leaves of C. creticus subsp. creticus. Histochemical staining of leaves’ trichomes and comprehensive phytochemical characterization of resin, leaves and their exudates, indicated that long-stalked capitate trichomes of C. creticus subsp. creticus, grown both in vitro (IV) and in greenhouse (GH), are capable of producing bioactive oleoresin-related terpenoids and phenylpropanoids compounds. Bioactivity-guided approach was implemented in search for the major antibacterial compound in C. creticus resin against two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and two Gram-positive bacteria (Bacillus cereus and Micrococcus flavus). Bioautographic assay on TLC plates with separated components of Cistus resin extract, revealed a pronounced zone of microbial growth inhibition, corresponded to a highly active compound with Rf values of 0.45, structurally characterized and identified as ent-3β-acetoxy-13-epi-manoyl oxide. This finding opens the route for focusing on isolation and functional characterization of genes involved in the biosynthesis of ent-3β-acetoxy-13-epi-manoyl oxide and its precursor ent-3β-hydroxy-13-epi-manoyl oxide, with the aim to establish sustainable in vitro biotechnological protocols for its large-scale production in homologous and heterologous plant and microbial hosts.
•Chemical profiles of differently grown C. creticus plants and resin are comparable.•Terpenoids and polyphenolics accumulate in the long-stalked capitate trichomes.•ent-3β-acetoxy-13-epi-manoyl oxide is the most potent antimicrobial compound.
The content and chemical composition of Micromeria pulegium (Rochel) Benth. essential oils were studied in native plant material at vegetative stage and in micropropagated plants, obtained from nodal ...segments cultured on solid MS medium supplemented with N⁶–benzyladenine (BA) or kinetin at different concentrations, alone or in combination with indole-3-acetic acid. Shoot proliferation was achieved in all treatments, but the highest biomass production was obtained after treatment with 10 µM BA. Phytochemical analysis identified up to 21 compounds in the essential oils of wild-growing and in vitro cultivated plants, both showing very high percentages of total monoterpenoids dominated by oxygenated monoterpenes of the menthane type. Pulegone and menthone were the main essential oil components detected in both wild-growing plants (60.07 and 26.85 %, respectively) and micropropagated plants grown on either plant growth regulator-free medium (44.57 and 29.14 %, respectively) or BA-supplemented medium (50.77 and 14.45 %, respectively). The percentage of total sesquiterpenoids increased in vitro, particularly owing to sesquiterpene hydrocarbons that were not found in wild-growing plants. Differences in both content and the composition of the essential oils obtained from different samples indicated that in vitro culture conditions and plant growth regulators significantly influence the essential oils properties. In addition, the morphology and structure of M. pulegium glandular trichomes in relation to the secretory process were characterized for the first time using SEM and light microscopy, and their secretion was histochemically analyzed.
We present changes in Tacitus bellus antioxidative system that specifically correspond to subsequent phases of hemibiotroph Fusarium verticillioides infection revealed by histological analysis. T. ...bellus response to spore germination 6 h post inoculation (hpi), manifested as first oxidative burst, was characterized by transient decrease in malondialdehyde (MDA) content, transient increase in catalase (CAT), low level of superoxide dismutase (SOD) and peroxidase (POD) activity, as well as with transient decrease in total antioxidant capacity (TAC), total phenol content (TPC) and phenylalanine ammonium lyase activity (PAL), and no changes in polyphenol oxidase (PPO) activity, or phenolic profile. During the biotrophic phase of F. verticillioides infection, characterized by hyphae spread intercellularly in epidermal and mesophyll tissue, the host antioxidative system was suppressed. The transition to necrotrophic phase of F. verticillioides infection (inter- and intracellular colonization and sporulation), occurred 3–4 days post inoculation (dpi). During the necrotrophic phase, 5–7 dpi, slowed progression of colonization of T. bellus mesophyll cells occurred and it coincided with sharp increase in MDA content and CAT, SOD and POD activities, but the drop in TAC, TPC content, and PPO activity, as well as the production of phytotoxin fusaric acid. Presented results add to the knowledge of events and mechanisms related to the transition from biotrophy to necrotrophy in F. verticillioides.
Unlike garlic and onion, the regeneration of chive (Allium schoenoprasum L.), cultivated both for culinary and ornamental purposes, has not been intensively studied. The effects of the eight ...cytokinins and the plant basal section thickness on regeneration efficiency and subsequent plant growth were studied. Representatives of all cytokinin structural groups: isoprenoide side chain (trans-zeatin) and aromatic side chain (benziladenine, kinetin, meta-topolin) adenine derivatives, and phenylurea derivatives (thidiazuron and N-(2-chloro-4-pyridyl)-N′-phenylurea) at 0, 1, 5 or 10 μM were used. Histological analysis revealed adventitious buds formation from the leaf sheaths’ bases and the basal plate. The highest regeneration frequency (100 %) and the mean bud number per explant (20.0) were achieved with 10 μM thidiazuron (TDZ), and 5 mm-thick basal sections were the most responsive explants. Inferior shoot and root growth characteristics of plants regenerated by this treatment was avoided by exclusion or replacement of 10 μM TDZ with 5 μM kinetin (Kin) after a 4-week bud induction period, without consequences on the regeneration efficiency. In addition, a positive correlation between peroxidase, catalase and superoxide dismutase activity and the regeneration capacity was observed. All antioxidative enzymes activity changed much faster with 10 μM TDZ than with 1 μM Kin, which provoked the weakest regeneration response. Moreover, a unique peroxidase isoform was observed only in TDZ-treated explants after 3rd day of treatment. This work is useful for genetic engineering and virus-free plant production advancement, and for the knowledge expansion regarding the role of antioxidative enzymes in plant organogenesis.
Leaf senescence is a highly regulated final phase of leaf development preceding massive cell death. It results in the coordinated degradation of macromolecules and the subsequent nutrient relocation ...to other plant parts. Very little is still known about early stages of leaf senescence during normal leaf ontogeny that is not triggered by stress factors. This paper comprises an integrated study of natural leaf senescence in tobacco plants grown in vitro, using molecular, structural, and physiological information. We determined the time sequence of ultrastructural changes in mesophyll cells during leaf senescence, showing that the degradation of chloroplast ultrastructure fully correlated with changes in chlorophyll content. The earliest degenerative changes in chloroplast ultrastructure coinciding with early chromatin condensation were observed already in mature green leaves. A continuum of degradative changes in chloroplast ultrastructure, chromatin condensation and aggregation, along with progressive decrease in cytoplasm organization and electron density were observed in the course of mesophyll cells ageing. Although the total amounts of endogenous cytokinins gradually increased during leaf ontogenesis, the proportion of bioactive cytokinin forms, as well as their phosphate precursors, in total cytokinin content rapidly declined with ageing. Endogenous indole-3-acetic acid (IAA) levels were strongly reduced in senescent leaves, and a decreasing tendency was also observed for abscisic acid (ABA) levels. Senescence-associated tobacco cysteine proteases (CP, E.C. 3.4.22) CP1 and CP23 genes were induced in the initial phase of senescence. Genes encoding glutamate dehydrogenase (GDH, E.C. 1.4.1.2) and one isoform of cytosolic glutamine synthetase (GS1, E.C. 6.3.1.2) were induced in the late stage of senescence, while chloroplastic GS (GS2) gene showed a continuous decrease with leaf ageing.
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