Andrzej Kaźmierczak

Determination of ACC-induced cell-programmed death in roots of Vicia faba ssp. minor seedlings by acridine orange and ethidium bromide staining

Fluorescence staining with acridine orange (AO) and ethidium bromide (EB) showed that nuclei of cortex root cells of 1-aminocyclopropane-1-carboxylic acid (ACC)-treated Vicia faba ssp. minor seedlings differed in color. Measurement of resultant fluorescence intensity (RFI) showed that it increased when the color of nuclear chromatin was changed from green to red, indicating that EB moved to the nuclei via the cell membrane which lost its integrity and stained nuclei red. AO/EB staining showed that changes in color of the nuclear chromatin were accompanied by DNA condensation, nuclei fragmentation, and chromatin degradation which were also shown after 4,6-diamidino-2-phenylindol staining. These results indicate that ACC induced programmed cell death. The increasing values of RFI together with the corresponding morphological changes of nuclear chromatin were the basis to prepare the standard curve; cells with green unchanged nuclear chromatin were alive while those with dark orange and bright red nuclei were dead. The cells with nuclei with green–yellow, yellow–orange, and bright orange chromatin with or without their condensation and fragmentation chromatin were dying. The prepared curve has became the basis to draw up the digital method for detection and determination of the number of living, dying, and dead cells in an in planta system and revealed that ACC induced death in about 20% of root cortex cells. This process was accompanied by increase in ion leakage, shortening of cells and whole roots, as well as by increase in weight and width of the apical part of roots and appearance of few aerenchymatic spaces while not by internucleosomal DNA degradation.

Effect of BAP and IAA on the expression of G1 and G2 control points and G1-S and G2-M transitions in root meristem cells of Vicia faba.

Excised, carbohydrate‐starved root meristems of Vicia faba subsp. minor have been used to investigate the impact of the auxin indole‐3‐acetic acid (IAA) and the cytokinin benzyl‐6‐aminopurine (BAP) on (1) the expression of Principal Control Points (PCPs) during the G1‐ and G2‐phases of the cell cycle, and (2) the dynamics of sucrose‐mediated resumption of DNA replication and mitosis (G1‐to‐S and G2‐to‐M transitions). Compared with the excised root tips starved in mineral medium without hormones, stationary phase meristems induced during continuous treatment with BAP, IAA, or a mixture of BAP+IAA, increased the number of G2 cells, producing characteristic profiles of nuclear DNA content. In medium containing 2% sucrose, BAP accelerated PCP1→S and PCP2→M, whereas continuous treatment with IAA resulted in marked prolongation of both transitions. Since the PCPs regulate progression through the key events of interphase and mitosis by interacting with cyclin dependent kinases (CDKs), these results seem to correspond with current data indicating functional connections between phytohormones, nutritional signals, gene expression and the cell division cycles in plants.

Cytokinins résumé: their signaling and role in programmed cell death in plants.

Cytokinins (CKs) are a large group of plant hormones which play a crucial role in many physiological processes in plants. One of the interesting functions of CKs is the control of programmed cell death (PCD). It seems that all CKs-dependent phenomena including PCD are accompanied by special multi-step phosphorelay signaling pathway. This pathway consists of three elements: histidine kinase receptors (HKs), histidine phosphotransfer proteins (HPs) and response regulators (RRs). This review shows the résumé of the latest knowledge about CKs signaling pathways in many physiological processes in plants with special attention paid to PCD process.

Kinetin induces cell death in root cortex cells of Vicia faba ssp. minor seedlings

The double fluorescence staining with acridine orange and ethidium bromide (AO/EB) revealed that treatment of Vicia faba ssp. minor seedlings with kinetin-induced programmed cell death (PCD) in root cortex cells. Kinetin-induced cell death reflected by the morphological changes of nuclei including their invagination, volume increase, chromatin condensation and degradation as well as formation of micronuclei showed by AO/EB and 4,6-diamidino-2-phenylindol staining was accompanied by changes including increase in conductivity of cell electrolytes secreted to culture media, decrease in the number of the G1- and G2-phase cells and appearance of fraction of hypoploid cells as the effect of DNA degradation without ladder formation. Decrease in the number of mitochondria and in the activity of cellular dehydrogenases, production of reactive oxygen species (ROS), appearance of small and then large lytic vacuoles and increase in the amount of cytosolic calcium ions were also observed. The PCD was also manifested by increased width and weight of apical fragments of roots as well as decreased length of cortex cells which led to shortening of the whole roots. The kinetin-induced PCD process was almost completely inhibited by adenine, an inhibitor of phosphoribosyl transferase, and mannitol, an inhibitor of ROS production. These cell-death hallmarks and pathway of this process suggested that the induction of kinetin-specific vacuolar type of death, expressed itself with similar intensity on both morphological and metabolic levels, was a transient protecting whole roots and whole seedlings against elimination.

The crucial elements of the ‘last step’ of programmed cell death induced by kinetin in root cortex of V. faba ssp. minor seedlings

Key messageKinetin-induced programmed cell death, manifested by condensation, degradation and methylation of DNA and fluctuation of kinase activities and ATP levels, is an autolytic and root cortex cell-specific process.AbstractThe last step of programmed cell death (PCD) induced by kinetin in the root cortex of V. faba ssp. minor seedlings was explained using morphologic (nuclear chromatin/aggregation) and metabolic (DNA degradation, DNA methylation and kinases activity) analyses. This step involves: (1) decrease in nuclear DNA content, (2) increase in the number of 4′,6-diamidino-2-phenylindole (DAPI)-stained chromocenters, and decrease in chromomycin A3 (CMA3)-stained chromocenters, (3) increase in fluorescence intensity of CMA3-stained chromocenters, (4) condensation of DAPI-stained and loosening of CMA3-stained chromatin, (5) fluctuation of the level of DNA methylation, (6) fluctuation of activities of exo-/endonucleolytic Zn2+ and Ca2+/Mg2+-dependent nucleases, (7) changes in H1 and core histone kinase activities and (8) decrease in cellular ATP amount. These results confirmed that kinetin-induced PCD was a specific process. Additionally, based on data presented in this paper (DNA condensation and ATP depletion) and previous studies [increase in vacuole, increase in amount of cytosolic calcium ions, ROS production and cytosol acidification “in Byczkowska et al. (Protoplasma 250:121–128, 2013)”], we propose that the process resembles autolytic type of cell death, the most common type of death during development of plants. Lastly, the observations also suggested that regulation of these processes might be under control of epigenetic (methylation/phosphorylation) mechanisms.

Determination of GA3 in Chara vulgaris by capillary electrophoresis system

Method for simultaneous measurement of gibberellic acid was applied using capillary zone electrophoresis. Gibberellic acid was identified in extracts of apical part of thallus of Chara vulgaris L. The amount of gibberellins measured on the basis of activity determined by the micro-drop bioassay (59.8 mg·kg−1; with gibberellic acid as a standard) was comparable with that estimated by capillary electrophoresis (54.9 mg·kg−1).

Cell cycle-dependent phosphorylation of pRb-like protein in root meristem cells of Vicia faba

The retinoblastoma tumor suppressor protein (pRb) regulates cell cycle progression by controlling the G1-to-S phase transition. As evidenced in mammals, pRb has three functionally distinct binding domains and interacts with a number of proteins including the E2F family of transcription factors, proteins with a conserved LxCxE motif (D-type cyclin), and c-Abl tyrosine kinase. CDK-mediated phosphorylation of pRb inhibits its ability to bind target proteins, thus enabling further progression of the cell cycle. As yet, the roles of pRb and pRb-binding factors have not been well characterized in plants. By using antibody which specifically recognizes phosphorylated serines (S807/811) in the c-Abl tyrosine kinase binding C-domain of human pRb, we provide evidence for the cell cycle-dependent changes in pRb-like proteins in root meristems cells of Vicia faba. An increased phosphorylation of this protein has been found correlated with the G1-to-S phase transition.

The level of endogenous 1-aminocyclopropane-1-carboxylic acid in gametophytes of Anemia phyllitidis is increased during GA3-induced antheridia formation

Capillary electrophoresis revealed that the endogenous level of ACC (1-aminocyclopropane-1-carboxylic acid) in the gametophytes of Anemia phyllitidis was elevated during GA3-induced male determination, whereas AOA (aminooxyacetic acid, specific inhibitor of ACC synthase) in untreated as well as in the GA3-treated gametophytes decreased concentration of ACC. The mechanism of ethylene involvement in controlling antheridiogenesis reflected at the level of ACC, which is supposed to mediate interactions between ethylene and gibberellins, is proposed.

Kinetin-induced programmed death of cortex cells is mediated by ethylene and calcium ions in roots of Vicia faba ssp. minor

Kinetin-induced programmed death of cortex cells made the roots of Vicia faba ssp. minor (faba bean) seedlings shorter and thicker since the respective cells were not as long but wider then in the untreated seedlings (control). Thus the weight of 2-cm long apical parts of the kinetin-treated roots was greater. Furthermore, apical hooks as well as aerenchyma formation in the roots were observed. The presented studies aimed at revealing the role of ethylene in kinetin-induced cell death. To this end ACC (1-aminocyclopropane-1-carboxylic acid; ethylene precursor) and calcium ion amounts were analysed. Influence of ethylene biosynthesis and reception inhibitors on viability of cells and on the amount and distribution of calcium ions was also assessed. The results of the studies showed that both calcium ions and ethylene were involved in the kinetin-induced cell death. This allowed us to discuss their crucial role in this process and to propose a general scheme of induction of aerenchyma formation pathway resulting from kinetin-induced death of cortex cells.

Membrane-related hallmarks of kinetin-induced PCD of root cortex cells

Key messageChanges in cellular membrane potential and their fluidisation are the hallmarks of cell death induction with kinetin in root cortex.AbstractProgrammed cell death (PCD), one of the essential processes in plant development, is still poorly understood. In this paper, the scientific plant model, V. faba ssp. minor seedling roots after kinetin application which triggers off programmed death of cortex cells, was used to recognise membrane-related aspects of plant cell death. Spectrophotometric, reflectometric and microscopic studies showed that the PCD induced by kinetin is accompanied by higher potassium ions leakage from roots, loss of plasma and ER membrane potentials (expressed by their lower amounts and higher index of fatty acid unsaturation), malformation of nuclear envelope, lower total lipid amount and formation of their peroxides, lower amount of phospholipids and changes in their composition. The results showed that potassium ions leakage, expressed in percentage of their amounts, and loss of plasma and ER membrane potential, expressed in percentage of their fluorescence intensity, together with the nuclear chromatin double staining with ethidium bromide and acridine orange, might be direct and universal methods for detecting specific plant PCD hallmarks and estimation of PCD intensity (percentage of dying and dead cells).