Coupling of Cell Division and Differentiation in Arabidopsis thaliana Cultured Cells with Interaction of Ethylene and ABA Signaling Pathways

Abstract

Recent studies indicate direct links between molecular cell cycle and cell differentiation machineries. Ethylene and abscisic acid (ABA) are known to affect cell division and differentiation, but the mechanisms of such effects are poorly understood. As ethylene and ABA signaling routes may interact, we examined their involvement in cell division and differentiation in cell tissue cultures derived from several Arabidopsis thaliana plants: wild type (Col-0), and ethylene-insensitive mutants etr1-1, ctr1-1, and ein2-1. We designed an experimental setup to analyze the growth-related parameters and molecular mechanisms in proliferating cells upon short exposure to ABA. Here, we provide evidence for the ethylene-ABA signaling pathways' interaction in the regulation of cell division and differentiation as follows: (1) when the ethylene signal transduction pathway is functionally active (Col-0), the cells actively proliferate, and exogenous ABA performs its function as an inhibitor of DNA synthesis and division; (2) if the ethylene signal is not perceived (etr1-1), then, in addition to cell differentiation (tracheary elements formation), cell death can occur. The addition of exogenous ABA can rescue the cells via increasing proliferation; (3) if the ethylene signal is perceived, but not transduced (ein2-1), then cell differentiation takes place-the latter is enhanced by exogenous ABA while cell proliferation is reduced; (4) when the signal transduction pathway is constitutively active, the cells begin to exit the cell cycle and proceed to endo-reduplication (ctr1-1). In this case, the addition of exogenous ABA promotes reactivation of cell division.

Keywords: Arabidopsis thaliana; abscisic acid; cell culture; cell differentiation; cell proliferation; ethylene.

Figure 1

The effect of abscisic acid (ABA) on the phenotype (a) and growth indexes (Is) (b) of suspension cultures of A. thaliana Col-0, etr1-1, and ein2-1. Cell cultures of three genotypes were grown in the medium supplemented with 50 µM ABA (+ABA) for 10 days. (–ABA): control tissue culture not supplemented with ABA. At the end of sub-culturing, Is values and number of TEs were determined. Values presented are means of three independent experiments with five replicates in each ± SE (standard error). Data are significant at p < 0.05.

Figure 2

ABA (25 µM) affects 5-bromo-2-deoxyuridine (BrdU) incorporation into the DNA of Col-0, etr1-1, ctr1-1, and ein2-1 cell lines. Genomic DNA (2 μg) was isolated from cultured cells and denatured. Single-stranded DNA was applied to Hybond-C using a Bio-Dot SF Microfiltration apparatus (a). BrdU incorporation into DNA was quantified (b) with monoclonal anti-BrdU antibodies (1:2000 dilution). For visualization, anti-mouse antibodies (dilution 1:7000) conjugated with horseradish peroxidase were used. Numbers in (b) correspond to relative BrdU signal intensities quantified as optical density of dot-blots.

Figure 3

Effect of U0126 protein kinase kinase (MKK) inhibitor on ABA-dependent MBP-phosphorylation of the soluble proteins isolated from Col-0, etr1-1, and ctr1-1 cultures. Soluble proteins were isolated from ABA/U0126-untreated cells and treated with both substances. Myelin basic protein (MBP) phosphorylation was tested in vitro with [γ-³²P]ATP. Radioautographic image is presented.

Figure 4

Expression of individual genes coding for MPKs in Col-0, etr1-1, and ein2-1 cultured cells after 3 h treatment with ABA. mRNA levels in cultured Col-0, etr1-1 and ein2-1 cells were examined by RT-PCR. For RT-PCR analysis, AtACT2 was used as a reference gene. Data on a typical experiment are presented.

Figure 5

Expression of individual genes coding for SnRK2s in Col-0, etr1-1 and ein2-1 cultures after 3 h treatment with ABA, estimated by RT-PCR with AtACT2 as a reference gene. Results of a typical experiment are presented.

Figure 6

The effect of ABA (25 µM) on the MBP phosphorylation in vitro (a) and in gel (in situ) (b) in protein fractions enriched with low-abundance proteins using a MicroRotofor IEF Cell. Radioautographic image is presented.

Figure 7

Radioautographic images of 2D-gels of the ABA-dependent phosphorylated proteins separated and enriched in the MicroRotofor IEF Cell. Fractions with pH 5.2 – 5.4 (a) and 6.2 – 6.8 (b) were used for 2D-gels (7 cm strips, pH 4.0–7.0). The proteins identified by MALDI-TF MS are numbered.

Figure 8

Schematic representation of the cellular events occurring in cultured Arabidopsis cells treated with ABA.