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

Abstract

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.

Fig. 1

Control (a) and kinetin-treated (b) seedlings of V. faba ssp. minor. Scale bars are 20 mm

Fig. 2

Microphotographs of the nuclei in living, dying and dead cells in the root cortex of V. faba ssp. minor seedlings after 46 μM kinetin treatment, detected by AO/EB staining in thin longitudinal sections of the zone I (a) and zone II (b) of root. c Green nuclei of living cells, green-yellow and green-yellow-orange nuclei of PCD cells with different forms of condensed (d, e) and disappearing (f, g) chromatin and bright red nuclei of dead cells (h). Arrows indicate PCD cells. Scale bars in a–b are 20 μm and in c–h are 10 μm

Fig. 3

The number of living, PCD-dying and dead cells in the root cortex V. faba ssp. minor seedlings of untreated (a) and treated with kinetin for 48, 72, 96 h (b–d) as well as of dying cells at early and late stage of PCD (b’–d’) and those treated for 72 h with adenine (e), adenine with kinetin (f), mannitol (g) and mannitol with kinetin (h). Error bars represent the SE of the mean of three independent experiments

Fig. 4

Microphotographs of the nuclei of kinetin-treated V. faba ssp. minor seedling root cells after DAPI showing the unchanged nuclei (a), nuclei with chromatin condensation (b), formation of micronuclei (c), nuclei invagination (d), disappearance of chromatin (e, f) and DNA fragmentation (g, h). Scale bar in “e” = 10 μm is applied to all figures

Fig. 5

Electropherogram of samples of DNA isolated from the apical part of kinetin-treated and untreated V. faba ssp. minor seedling roots and DNA markers (1,550–10,000 bp)

Fig. 6

Histograms (a–d) displaying the percentage frequency distribution of cells (a’–d’) in the G1- (peak around 9 a.u.), G2- (peak around 19 a.u.) and S- (represented as the gaps between the peaks for phases G1 and G2), endoreplication- (>4C DNA) phases and hypoploid fraction (<2C) indicated by an arrow, microcytophotometrically determined in the zone I (a, a’, c, c’) and II (b, b’, d, d’) of untreated (a, a’, b, b’) and kinetin-treated (c, c’, d, d’) V. faba ssp. minor seedling roots. Error bars (a’–d’) represent the SE of the mean of three independent experiments

Fig. 7

The number of mitochondria in the zone II of mid parts cortex cells of the untreated (a1) and kinetin-treated roots (a2) and activity of cellular dehydrogenases secreted (b) from the zone I and II of untreated and kinetin-treated V. faba ssp. minor seedling roots. Scale bars are 50 μm. Error bars represent the SE of the mean of three independent experiments

Fig. 8

Microphotographs of ROS production (a, b, b’; dark arrows), formation of small (c, d, d’; dark arrows) and large (e) lytic vacuoles, calcium ion distribution (f, g, white arrows) and its amount (h) expressed in arbitrary units (a.u.) of fluorescence intensity of the zone II of untreated (a, c, f, h) and kinetin-treated (b, b’, d, d’, e, g, h) V. faba ssp. minor seedling roots. Scale bar in a is 100 μm, in b is 50 μm, in b’ and c, d, e, f, g is 10 μm and in d’ is 20 μm. Error bars in the “i” figure represent the SE of the mean of three independent experiments