Genome-wide analysis of gene expression profiles associated with cell cycle transitions in growing organs of Arabidopsis

Abstract

Organ growth results from the progression of component cells through subsequent phases of proliferation and expansion before reaching maturity. We combined kinematic analysis, flowcytometry, and microarray analysis to characterize cell cycle regulation during the growth process of leaves 1 and 2 of Arabidopsis (Arabidopsis thaliana). Kinematic analysis showed that the epidermis proliferates until day 12; thereafter, cells expand until day 19 when leaves reach maturity. Flowcytometry revealed that endoreduplication occurs from the time cell division rates decline until the end of cell expansion. Analysis of 10 time points with a 6k-cDNA microarray showed that transitions between the growth stages were closely reflected in the mRNA expression data. Subsequent genome-wide microarray analysis on the three main stages allowed us to categorize known cell cycle genes into three major classes: constitutively expressed, proliferative, and inhibitory. Comparison with published expression data obtained from root zones corresponding to similar developmental stages and from synchronized cell cultures supported this categorization and enabled us to identify a high confidence set of 131 proliferation genes. Most of those had an M phase-dependent expression pattern and, in addition to many known cell cycle-related genes, there were at least 90 that were unknown or previously not associated with proliferation.

Figure 1.

Kinematic analysis of leaf growth. A, Leaf area. B, Relative leaf expansion rate (RLER). C, Cell area. D, Number of cells per leaf. E, Cell division rate. F, Stomatal index. Symbols denote averages ± se of three replicate experiments. At the bottom the growth phases are indicated proliferation (cells divide and expand simultaneously), expansion (expansion in absence of division), and mature (no more cell growth).

Figure 2.

Flow cytometry analysis of nuclear DNA content. The results of a representative experiment are plotted. The bar at the bottom denotes the growth phases based on the kinematic analysis (Fig. 1).

Figure 3.

Clustering of developmental time series support tree analysis (Graur and Li, 2000) of the expression data of 2,061 significantly modulated genes (P ≤ 0.001) comparing time points. The level of support for each branch of the tree is color coded as indicated in the legend; the level of support indicates the confidence for each branch. The bar on the right denotes the growth phases based on the kinematic analysis (Fig. 1).

Figure 4.

Clustering of gene expression profiles by QT-Clust analysis (Heyer et al., 1999) of the expression profiles of 2,061 significantly modulated genes (P ≤ 0.001). Cluster number and size are indicated. The abscissa denotes the time after sowing, which is enlarged for Cluster 14, and the ordinate indicates normalized and median-centered expression levels. The colored bar shows the corresponding growth phases based on the kinematic analysis (Fig. 1).

Figure 5.

Enrichment of cell cycle genes among those that show modulation during leaf development, in different developmental zones of the root tip and in synchronized cell cultures. The distribution of all genes on the array is compared to that of core cell cycle genes defined by Vandepoele et al. (2002) and Wang et al. (2004). Numbers subsequently indicate total number of genes in a population; the number of core cell cycle genes and the percentage of genes that are core cell cycle genes (in parentheses).