Concentration-dependent effects of narciclasine on cell cycle progression in Arabidopsis root tips

Abstract

Background: Narciclasine (NCS) is an Amaryllidaceae alkaloid isolated from Narcissus tazetta bulbs. NCS has inhibitory effects on a broad range of biological activities and thus has various potential practical applications. Here we examine how NCS represses plant root growth.

Results: Results showed that the inhibition of NCS on cell division in Arabidopsis root tips and its effects on cell differentiation are concentration-dependent; at low concentrations (0.5 and 1.0 μM) NCS preferentially targets mitotic cell cycle specific/cyclin complexes, whereas at high concentration (5.0 μM) the NCS-stimulated accumulation of Kip-related proteins (KRP1 and RP2) affects the CDK complexes with a role at both G1/S and G2/M phases.

Conclusions: Our findings suggest that NCS modulates the coordination between cell division and differentiation in Arabidopsis root tips and hence affects the postembryonic development of Arabidopsis seedlings.

Figure 1

Effects of NCS on the post-embryonic development of Arabidopsis roots. (A) Inhibitory effect of NCS on radicle protrusion during seed germination. (B) Wild type Arabidopsis seedling at 10 days after stratification grown on medium containing 0 or 5.0 μM NCS. Bar = 5 mm. (C) A close-up view of the whole plant in (B) treated with 5.0 μM NCS. Bar = 1 mm. (D) NCS inhibits primary root elongation. Root growth time course was measured after transferring 3-day-old Arabidopsis seedlings to NCS-containing medium. (E) NCS affects the lateral root density after exposing to NCS for 7 days. Data shown in (A), (D), (E) are means ± SD obtained from three independent experiments (n ≧ 20, P < 0.01). (F) Effects of NCS on cell expansion and cell division of meristematic cells. Arrows in the control panel showed the dividing cells. Bars = 50 μm in all panels. Differential interference contrast (DIC) images were captured using the transmission light detector of the confocal microscope.

Figure 2

Effects of NCS on cell division and cell differentiation in Arabidopsis root tips. (A) NCS affects the differentiation of xylem pole pericycle cells in the root tip. Bar = 200 μm. (B) The development of root hairs in Arabidopsis primary roots was affected by NCS. Data shown are means ± SD obtained from three independent experiments (n ≧ 20). (C) CDKA;1::uidA promoter activity after NCS treatment for 4 days. Bar = 200 μm. (D) Expression of the mitotic marker CYCB1;1::uidA after 2 days NCS treatment. Bar = 50 μm.

Figure 3

Using a tobacco cell suspension to determine the effects of NCS on cell cycle. (A) The phenotype of 7 days after germination (DAG) tobacco seedlings grown on 0.5 μM NCS. Bar = 5 mm. (B) Primary root length of 7 DAG tobacco seedlings was inhibited by NCS. Values are means ± SD of three independent experiments (n ≧ 20). (C) Images of BY-2 cells treated with NCS and depletion of 2,4-D for 2 days. Bar = 50 μm. (D) Flow cytometric analysis of the cell cycle. (E) qRT-PCR detection of CYCD3;1, Histone H4, CYCA1;1 and CYCB1;1 mRNA after NCS-treatment in synchronized tobacco BY-2 cells. (F) E2Fa and RBR transcription level in BY-2 cells after NCS-treatment. The relative expression is normalized to ACTIN2. Data shown were the means ± S.D of three independent experiments.

Figure 4

qRT-PCR analysis of the expression of core cell cycle genes in Arabidopsis root tips. (A) qRT-PCR results showing the effects of NCS on expression of CDKA;1, CYCD3;1, CYCD3;2, E2Fa, CDKB1;1 and CDKB2;1 in Arabidopsis root tips. (B) The relative expression (normalized to PP2A) of DNA polymerase α and CDC6a after NCS treatment for indicated times. Data shown in (A) and (B) are means ± SD of three independent experiments.

Figure 5

Analysis of KRP1 and 2 expression and CDK activity after NCS treatment. (A) qRT-PCR analysis of KRP1 and KRP2 expression relative to PP2A. (B) Immunoblot analysis of NCS-treated Arabidopsis roots using an anti-KRP2 antibody. Coomassie Brilliant Blue staining of the electrophoresis gel was used as a loading control. (C) P10^CKS1At-associated kinase activity in NCS-treated roots. Autoradiogram showing typical results of CDK activity assays with histone as the substrate. Coomassie Brilliant Blue staining of the electrophoresis gel with histone was used as a control for equal substrate quantity per phosphorylation reaction. (D) Relative quantification of three independent kinase activity measurements as depicted in (C). The control was arbitrarily set at 100%. Data shown in (A) and (D) are means ± SD of three independent experiments.