Transcriptional profile of the Arabidopsis root quiescent center

Abstract

The self-renewal characteristics of stem cells render them vital engines of development. To better understand the molecular mechanisms that determine the properties of stem cells, transcript profiling was conducted on quiescent center (QC) cells from the Arabidopsis thaliana root meristem. The AGAMOUS-LIKE 42 (AGL42) gene, which encodes a MADS box transcription factor whose expression is enriched in the QC, was used to mark these cells. RNA was isolated from sorted cells, labeled, and hybridized to Affymetrix microarrays. Comparisons with digital in situ expression profiles of surrounding tissues identified a set of genes enriched in the QC. Promoter regions from a subset of transcription factors identified as enriched in the QC conferred expression in the QC. These studies demonstrated that it is possible to successfully isolate and profile a rare cell type in the plant. Mutations in all enriched transcription factor genes including AGL42 exhibited no detectable root phenotype, raising the possibility of a high degree of functional redundancy in the QC.

Figure 1.

ET433 Enhancer Trap Expression. (A) Scheme of the Arabidopsis root tip. Tissues are depicted in a median longitudinal section with corresponding initials in lighter color at the base of each cell file. Epidermis (orange), cortex (yellow), endodermis (green), and stele (red) make up most of the root, and lateral (turquoise) and columella (blue) root caps surround the apex. Epidermis and lateral root cap share a common initial, as do endodermis and cortex. Initial cells surround the QC (white). (B) ET433 staining in a lateral root tip after emergence from the primary root. Pattern is identical in primary root tips at 7 d after germination. Arrowheads indicate QC cells.

Figure 2.

AGL42 cis Elements Mark the QC for Cell Sorting. (A) AGL42 genomic region (top), indicating exons (closed boxes) with corresponding protein-coding domains above, 5′ and 3′ UTRs (open boxes), and ET433 enhancer trap insertion site and orientation in the first intron. Promoter (construct I), partial first intron (construct II), and promoter plus intron (construct III) fusions to GFP are indicated with corresponding lengths of promoter and intron sequence. Note that terminator sequences (light green) were not derived from AGL42. MADS, MADS DNA binding domain; I, intervening region; K, K domain; C, C-terminal domain; TL, signal sequence for endoplasmic reticulum; block arrow, −46 minimal promoter from Cauliflower mosaic virus 35S promoter used in ET433. (B) Overlay of GFP (green) and propidium iodide (red) channels of construct III imaged at 4 d after germination. The inset shows the GFP channel only. (C) Same as (B) at 7 d after germination. (D) Fluorescence-activated cell sorter acquisition dot plots. Each dot corresponds to a single sorting event, and only cells with a high ratio of green to orange fluorescence (within the trapezoidal R4 gate) are collected. Background protoplasts show nearly equivalent levels of orange and green autofluorescence.

Figure 3.

Verification of the AGL42:GFP Transcriptional Profile. (A) Digital in situ of genes with known QC expression. Normalized expression values are shown for the QC (blue) and other root tissues. Expression in the QC is either significantly enriched over other tissues (red) or not (yellow). CRC, columella root cap; LRC, lateral root cap. See text for gene abbreviations. (B) Expression of a GFP fusion to the promoter of C2H2 basic domain/leucine zipper transcription factor AtWIP4 (At3g20880), predicted to be enriched in the QC. Note the strongest expression in the QC.

Figure 4.

Expression of Floral Regulators in the QC. (A) and (B) NAP promoter fusion to GFP exhibits some stele expression and enrichment in the QC. (C) PI promoter fusion to GFP has highest levels in the QC and young ground tissue. (D) PAN promoter fusion to GFP has highest expression in the columella initials and QC.

Figure 5.

Mutant Analysis and Expression of AGL42. (A) Genomic region of AGL42, indicating positions of insertions (wedges) and point mutations (asterisks) for corresponding allele numbers. agl42-4 is a splice acceptor mutation for the third exon, and agl42-5 gives rise to a G113R substitution in the fourth exon. Below is a scheme of the complete promoter and intron, with exon 1 bearing mutated ATGs (small asterisks) driving the GUS reporter (construct IV). (B) Whole-mount staining of construct IV at 7 d after germination showing tight QC expression. (C) Real-time quantitative RT-PCR of AGL42 transcript in whole roots as ratios relative to wild-type Columbia (Col) root. RNAi, RNA interference; Ws, Wassilewskija.

Figure 6.

Digital in Situ of Shoot Stem Cell Signaling Genes. CLV1 family of LRR receptor-like kinases. Normalized expression values are shown for the QC (blue) and other root tissues. Expression in the QC is either significantly enriched over other tissues (red) or not (yellow). CRC, columella root cap; LRC, lateral root cap.