Kinetin induces microtubular breakdown, cell cycle arrest and programmed cell death in tobacco BY-2 cells

Abstract

Plant cells can undergo regulated cell death in response to exogenous factors (often in a stress context), but also as regular element of development (often regulated by phytohormones). The cellular aspects of these death responses differ, which implies that the early signalling must be different. We use cytokinin-induced programmed cell death as paradigm to get insight into the role of the cytoskeleton for the regulation of developmentally induced cell death, using tobacco BY-2 cells as experimental model. We show that this PCD in response to kinetin correlates with an arrest of the cell cycle, a deregulation of DNA replication, a loss of plasma membrane integrity, a subsequent permeabilisation of the nuclear envelope, an increase of cytosolic calcium correlated with calcium depletion in the culture medium, an increase of callose deposition and the loss of microtubule and actin integrity. We discuss these findings in the context of a working model, where kinetin, mediated by calcium, causes the breakdown of the cytoskeleton, which, either by release of executing proteins or by mitotic catastrophe, will result in PCD.

Keywords: Callose; Cell cycle arrest; Kinetin; Microtubules; Programmed cell death; Tobacco BY-2.

Fig. 1

Physiological responses of tobacco BY-2 cells to kinetin (50 µM), added at day 3 after subcultivation. Time courses of A fresh weight of cells, B calcium, C glucose, D sucrose concentration in the medium and E conductivity of the medium, and their respective curve regression, R² values and P (A′–E′) indicating statistical significance between Ctrl and Kin series are shown. Data represent mean values and SE from three independent biological replications

Fig. 2

Cellular response of tobacco BY-2 cells to kinetin (50 µM), added at day 3 after subcultivation. A Time course of mortality for kinetin treatment from day 0 (Kin, d0), or from day 3 (Kin, d3) after subcultivation as compared to the untreated control (con). Data represent mean value from three independent experimental series scoring 600 individual cells per replication. B Representative cells stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) and viewed either at the peak of mitotic activity (day 3 after subcultivation) or at the time of maximal cell expansion (day 5 after subcultivation) either in control cells or cells treated with kinetin from day 0

Fig. 3

Frequency distributions over DNA content per nucleus inferred from DAPI staining through the cultivation cycle in control cells (A) and in cells that had been treated with 50 µM kinetin from day 0 (B). The DNA contents (C) were classified into hypoploid cells (< 2C), cells in G₁ phase (2C), cells in S phase (2–4C), cells in G₂ phase (4C) and endopolyploid cells (> 4C). Data represent mean and standard errors from two independent experiments consisting of three technical replications with a 600–800 individual nuclei per replication

Fig. 4

Appearance of BY-2 after double staining with Acridine Orange (membrane permeable green signal) and Ethidium Bromide (membrane impermeable, red signal) in untreated controls, or addition of 50 µM kinetin at the time of subcultivation. A Overview of the cells at day 3 or 7, respectively, merging the signals from the green and red channels. B Cellular details of the staging system used for classification in Fig. 5. nco nucleolus

Fig. 5

Frequency distributions over different stages of cell death as classified by double staining with Acridine Orange and Ethidium Bromide through the cultivation cycle in control cells (A) and in cells that had been treated with 50 µM kinetin from day 0 (B). The classification followed the system shown in Fig. 3B. Data represent mean and standard errors from two independent experiments consisting of three technical replications with 350–400 individual cells per replication

Fig. 6

Effect of kinetin on intracellular calcium levels reported by chloro-tetracycline. Representative cells are shown in A, either at the peak of mitotic activity (day 3 after subcultivation) or at the end of the cultivation cycle (day 7 after subcultivation) either in control cells or cells treated with 50 µM kinetin from day 0. Quantification of the fluorescent signal in whole cells (B) and in nuclei (C). Data represent mean and standard errors from two independent experiments consisting of three technical replications with 50 to 100 individual cells and nuclei per replication

Fig. 7

Effect of kinetin on callose abundance at the cross wall visualised by Aniline Blue. A Quantification of fluorescence in control cells from day 0 or in cells treated with 50 µM kinetin from day 3. Data represent mean and standard errors from two independent experiments consisting of three technical replications with a 150–250 individual cells per replication. B Representative images of the callose signal in control cells and C in kinetin-treated cells at day 7 after subcultivation. Heatmaps of signal abundance of callose, as yellow and red colour indicated by arrows, are shown in B′ and C′, respectively. cw, cell wall

Fig. 8

Graphical representation of Pearson correlation coefficients between cytosolic calcium (Ca²⁺), accumulation of callose, the different stages of cell death and the nuclear DNA content in response to kinetin in tobacco BY-2 cells

Fig. 9

Effect of kinetin on microtubules visualised by the marker TuB6-GFP and followed by spinning-disc confocal microscopy through days 3 until 7. Fifty-micromolar kinetin was added at day 3. Scale bars 10 µm. The situation at day 3 prior to addition of kinetin is shown in A-A″. B–E Control cells and B′–E′ and B″–E″ kinetin-treated cells

Fig. 10

Effect of kinetin on actin filaments visualised by the marker FABD2-GFP and followed by spinning-disc confocal microscopy through days 3 until 7. Fifty-micromolar kinetin was added at day 3. Scale bar in A = 10 µm is applied to all images. A and A″ The situation at day 3 prior to addition of kinetin in. B–E Control cells and B′–E′ and B″–E″ kinetin-treated cells