Transcript profiling of early lateral root initiation

Abstract

At the onset of lateral root initiation in Arabidopsis thaliana, the phytohormone auxin activates xylem pole pericycle cells for asymmetric cell division. However, the molecular events leading from auxin to lateral root initiation are poorly understood, in part because the few responsive cells in the process are embedded in the root and are thus difficult to access. A lateral root induction system, in which most xylem pole pericycle cells were synchronously activated by auxin transport inhibition followed by auxin application, was used for microarray transcript profiling. Of 4,600 genes analyzed, 906 significantly differentially regulated genes were identified that could be grouped into six major clusters. Basically, three major patterns were discerned representing induced, repressed, and transiently expressed genes. Analysis of the coregulated genes, which were specific for each time point, provided new insight into the molecular regulation and signal transduction preceding lateral root initiation in Arabidopsis. The reproducible expression profiles during a time course allowed us to define four stages that precede the cell division in the pericycle. These early stages were characterized by G1 cell cycle block, auxin perception, and signal transduction, followed by progression over G1/S transition and G2/M transition. All these processes took place within 6 h after transfer from N-1-naphthylphthalamic acid to 1-naphthalene acetic acid. These results indicate that this lateral root induction system represents a unique synchronized system that allows the systematic study of the developmental program upstream of the cell cycle activation during lateral root initiation.

Fig. 1.

(A) Schematic presentation of the sampling for transmission electron microscopy (TEM). The scissors indicate the position where the sections were taken. The highlighted box on the scheme shows the positioning of the micrographs represented in B and C.(B and C) TEM micrographs of transverse sections of NPA-(B) and NAA-treated (C) Arabidopsis roots. Note the strongly vacuolated cells after NPA treatment (B) and the appearance of dense cytoplasm after transfer to NAA (C) in the pericycle at the protoxylem poles. Pe, xylem pole pericycle cells; PX, protoxylem (arrows).

Fig. 2.

(A) Blot of-Log10 of the significance of the replicate effect versus the-Log10 of the significance of the time effect, illustrating the negative correlation between the significance of both effects. (B-G) Validation of reproducibility of the hybridizations by comparing the hybridization results of six genes for which two independent cDNA clones had been spotted on the array. (B) Unknown protein (GenBank accession no. Atg49740). (C) Flavonol sulfotransferase (At1g74090). (D) Putative protein (At3g5700). (E) Sedoheptulosebisphosphatase precursor (At3g55800). (F) Unknown protein (At3g07390). (H) ATP-sulfurylase precursor (At5g4370). (G) Comparison of the expression patterns of five cell cycle genes in LRIS by RT-PCR and microarray. Expression profiles of G₁/S cell cycle marker genes Arath;E2Fa, histone H4, and Arath; KRP2, and the G₂/M-specific genes Arath;CDKB1;1 and Arath;CYCB2;1.

Fig. 3.

(A) Hierarchical average linkage clustering. Clusters a, b, c, d, e, and f were induced at 2 h, 4 h, 6 h, transiently, down-regulated at 4 h, and at 2 h, respectively. (B) The six major clusters obtained by adaptive quality-based algorithm, representing up-regulated (1-3), transient (4), and down-regulated (5, 6) clusters.