Identification of transcription factors linked to cell cycle regulation in Arabidopsis

Abstract

Cell cycle is an essential process in growth and development of living organisms consists of the replication and mitotic phases separated by 2 gap phases; G1 and G2. It is tightly controlled at the molecular level and especially at the level of transcription. Precise regulation of the cell cycle is of central significance for plant growth and development and transcription factors are global regulators of gene expression playing essential roles in cell cycle regulation. This study has uncovered TFs that are involved in the control of cell cycle progression. With the aid of multi-parallel quantitative RT-PCR, the expression changes of 1880 TFs represented in the Arabidopsis TF platform was monitored in Arabidopsis synchronous MM2d cells during a 19 h period representing different time points corresponding to the 4 cell cycle phases after treatment of MM2d cells with Aphidicolin. Comparative TF expression analyses performed on synchronous cells resulted in the identification of 239 TFs differentially expressed during the cell cycle, while about one third of TFs were constitutively expressed through all time points. Phase-specific TFs were also identified.

Keywords: Arabidopsis; QRT-PCR; aphidicolin; cell cycle; suspension culture; synchronization; transcription factors.

Figure 1.

Growth pattern of the MM2d cell line (left) and early stationary phase (D5) (right). Cells were sub-cultured and cell growth of cell lines MM2d monitored by determination of the number of cells (×10⁵) per mL culture medium by hemocytometer.

Figure 2.

Flocytometry resault shows that majority of the cells proceed synchronous through S phase with an S-phase peak of 74% immediately after release of Aphidicolin.

Figure 3.

Microscopic observations. Peak of the M/AI was reached to about 12% in 10h after removal of Aphidicolin corresponding to M phase.

Figure 4.

Hierarchical clustering of cell cycle- relaated genes including 69 core cell cycle genes and 81 transcription factors. The fold change values for each sample relative to time zero as control were log₂ transformed. The mean values for the 3 independent biological replications were subjected to complete linkage hierarchical clustering. The color saturation reflects the magnitude of the averaged log₂FC. Expression values higher and lower than those of the control are shown in red and green respectively. The vertical dendrogram (left) indicates the relationship among the genes regarding their expression patterns. The color scale (top) indicates the color assigned to each log₂FC. The pattern has been created using MeV software ver.4.3.

Figure 5.

Expression profiles of cell cycle- related genes including 69 core cell cycle genes and 81 transcription factors. Cell cycle-regulated gene expression profiles are plotted against the different time points after removal of Aphidicolin. The pattern has been created using MeV software ver.4.3.

Figure 6.

Expression patterns of several cell cycle-related genes in Aphidicolin-induced synchrony (A-K). The fold change values for each sample relative to time zero as control were log₂ transformed. The mean values for the 3 independent biological replications were plotted as Log₂FC.

Figure 7.

The expression patterns of some constitutively expressed TFs during cell cycle progression.