Cell cycle progression in the pericycle is not sufficient for SOLITARY ROOT/IAA14-mediated lateral root initiation in Arabidopsis thaliana - PubMed (original) (raw)

. 2005 Nov;17(11):3035-50.

doi: 10.1105/tpc.105.035493. Epub 2005 Oct 21.

Bert De Rybel, Gerrit T S Beemster, Karin Ljung, Ive De Smet, Gert Van Isterdael, Mirande Naudts, Ryusuke Iida, Wilhelm Gruissem, Masao Tasaka, Dirk Inzé, Hidehiro Fukaki, Tom Beeckman

Affiliations

Cell cycle progression in the pericycle is not sufficient for SOLITARY ROOT/IAA14-mediated lateral root initiation in Arabidopsis thaliana

Steffen Vanneste et al. Plant Cell. 2005 Nov.

Abstract

To study the mechanisms behind auxin-induced cell division, lateral root initiation was used as a model system. By means of microarray analysis, genome-wide transcriptional changes were monitored during the early steps of lateral root initiation. Inclusion of the dominant auxin signaling mutant solitary root1 (slr1) identified genes involved in lateral root initiation that act downstream of the auxin/indole-3-acetic acid (AUX/IAA) signaling pathway. Interestingly, key components of the cell cycle machinery were strongly defective in slr1, suggesting a direct link between AUX/IAA signaling and core cell cycle regulation. However, induction of the cell cycle in the mutant background by overexpression of the D-type cyclin (CYCD3;1) was able to trigger complete rounds of cell division in the pericycle that did not result in lateral root formation. Therefore, lateral root initiation can only take place when cell cycle activation is accompanied by cell fate respecification of pericycle cells. The microarray data also yielded evidence for the existence of both negative and positive feedback mechanisms that regulate auxin homeostasis and signal transduction in the pericycle, thereby fine-tuning the process of lateral root initiation.

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Figures

Figure 1.

Figure 1.

Expression Analysis in the Wild Type and slr1. (A) to (D) Anatomical sections of PIAA14:GUS mature root tissue (A), detail of stele (B), elongation zone (C), and root meristem (D) in wild-type background. (E) and (F) PIAA14:GUS expression in root apical meristem in the wild type and in slr1, respectively. (G) and (H) Xylem pole pericycle-specific green fluorescent protein expression in mature root segment of J0121 in the wild type and in slr1, respectively. (I) and (J) PCDKA;1:GUS expression in the wild type and in slr1, respectively. (K) to (P) Expression in roots germinated on 10 μM NPA and transferred 72 h after germination to 10 μM NAA for 12 h of PDR5:GUS expression in the wild type (K), PDR5:GUS expression in slr1 (L), PCYCB1;1:GUS in the wild type (M), PCYCB1;1:GUS in slr1 (N), PCDKB1;1:GUS in the wild type (O), and PCDKB1;1:GUS in slr1 (P). C, cortex; En, endodermis; Ep, epidermis; L, lateral root cap; p, pericycle. Asterisk indicates protoxylem cells.

Figure 2.

Figure 2.

Schematic Representation of the LRIS. Seeds are germinated on medium supplemented with NPA to inhibit lateral root initiation. The pericycle cells of seedlings germinated on NPA are in G1 phase (0 h). Subsequent transfer to NAA-supplemented medium induces gradual cell cycle progression over S, G2, and M phases, corresponding to synchronized lateral root initiation. PCYCB1;1:GUS activity marks G2-to-M transition. Red rectangles indicate time points used for the microarray. The segment between root tip and root-hypocotyl junction (black lines at 0 h) was used for the microarray analysis.

Figure 3.

Figure 3.

Cluster Analysis of the Expression Data. (A) Schematic representation of the cross-table clustering methodology. A two-series data set (wild type and slr1) of 3110 profiles is combined into a single-series data set corresponding to 6220 profiles irrespective of genotypic background. This data set was clustered into 14 clusters and includes the major patterns within the single-series data set. Subsequently, the clusters were plotted in a cross-table format: the wild-type and slr1 expression clusters were plotted in front of the rows and at the head of the columns, respectively. All expression profiles over the different series are summarized by 142 (196) cluster combinations. (B) Clusters illustrating the major patterns of the combined data set. In a white–gray gradient, the time points of the single series are shown. Each time point is characterized by the individual biological repeated values (0 h = 72 h NPA, 2 h = 0 h + 2 h NAA, and 6 h = 0 h + 6 h NAA). Clusters 1 to 6, 7 to 11, and 12 to 14 correspond to upregulated, constitutive, and downregulated expression profiles, respectively. (C) Cross-table representation of the expression profiles within both genotypes. The frequencies of each cluster combination within the data set are indicated in each square. Gray marks no significant difference in induction rates between both genotypes, whereas red and orange and green and blue indicate that the induction rates are higher and lower in the wild type than in slr1, respectively. The black line outlines the selected 913 LRI genes.

Figure 4.

Figure 4.

Overrepresented Functional Categories within the SLR/IAA14-Mediated Upregulated Genes. For each significantly overrepresented functional category, the black and white bars represent the percentage of annotated genes within the 3110 significant genes versus all genes and within the 913 LRI genes versus the 3110 significant genes, respectively. Ordinate is 100%. The white bars illustrate the proportional enrichment of the genes in the functional categories when the selection is reduced from 3110 significant genes to 913 LRI genes.

Figure 5.

Figure 5.

Analysis of Primary Auxin Responsiveness of CYCD3;2, CYCA2;4, and CDKB2;1. (A) AREs within the 1000-bp sequence upstream of the 5′ untranslated region of CYCD3;2, CYCA2;4, and CDKB2;1. (B) Ratios of mean expression levels for CYCD3;2, CYCA2;4, and CDKB2;1 between CHX treatment and corresponding 0.5× Murashige and Skoog mock treatment for 2 and 4 h. The single asterisk and double asterisks indicate a significant difference between CHX-treated and mock-treated samples at P < 0.05 and P < 0.005, respectively.

Figure 6.

Figure 6.

Complementation of Cell Cycle Defect in slr1. (A) to (H) Overview of root phenotypes of 5-d-old seedlings of Col-0 (A), E2Fa/DPaOE (B), Col-0 × slr1 (C), E2Fa/DPaOE × slr1 (D), L_er_ (E), CYCD3;1OE (F), L_er_ × slr1 (G), and CYCD3;1OE × slr1 (H). (I) to (L) Microscopic analysis of the pericycle after clearing of mature L_er_ pericycle cell (I), L_er_ stage I lateral root primordium (J), mature L_er_ × slr1 pericycle cell (K), and zone of shortened pericycle cells in CYCD3;1OE × slr1 (L). Arrowheads mark pericycle cell size. (M) to (P) Real-time PCR analysis on L_er_, L_er_ × slr1, and CYCD3;1OE × slr1 roots of CYCA2;4 (M), CYCB1;1 (N), CYCB2;5 (O), and PLT1 (P). The single asterisk and double asterisks indicate significant reduction of expression compared with L_er_ at P < 0.01 and P < 0.001, respectively.

Figure 7.

Figure 7.

Analysis of Auxin Content in Col-0 and slr1. (A) IAA concentration of Col-0 and slr1 of the 3-mm most apical part of the primary root. Standard deviations are indicated by error bars. (B) and (C) PDR5:GUS expression in 5-d-old root apical meristems in Col-0 and slr1, respectively. (D) and (E) Anatomical analysis of PDR5:GUS in slr1 of the elongation zone and the root apical meristem, respectively. p, pericycle. The asterisks indicate protoxylem cells.

Figure 8.

Figure 8.

Model of SLR/IAA14-Dependent Lateral Root Initiation. (A) Induction of lateral root initiation (LRI) in the pericycle, resulting in a lateral root initiation site (LRS) when the auxin signaling cascade is intact. Further development toward a lateral root primordium is also dependent on auxin. When the polar auxin transport machinery is intact, auxin gradients are set up to organize the lateral root primordium (LRP). Disturbing the polar auxin transport (PAT) does not inhibit the auxin-induced developmental program of lateral root formation but provokes, rather, an unorganized multilayered proliferating zone (MLP) (Benková et al., 2003; Geldner et al., 2004). When the auxin signaling cascade is defective (slr1), no lateral roots can be initiated. Complementation of the cell cycle defect in slr1 by overexpression of CYCD3;1 is not sufficient to activate the developmental program of LRI; nevertheless, it can induce some proliferative divisions in the pericycle, resulting in a single-layered proliferating zone (SLP). (B) Model for AUX/IAA-mediated lateral root initiation (LRI). Black path: in the early auxin signaling cascade, increased auxin levels stimulate the degradation of AUX/IAA proteins, such as SLR/IAA14. AUX/IAA proteins repress the transcriptional activity of one or more ARFs. Downstream of this signaling cascade lies the activation of the developmental program of lateral root initiation that includes the coordinated action of cell fate respecification and cell cycle progression. Red paths: the auxin signaling cascade induces auxin conjugation and may negatively influence auxin content. Also, increased auxin levels induce AUX/IAA proteins that repress ARF transcriptional activity. Green paths: increased auxin levels can induce auxin transport, leading to even higher auxin levels. Furthermore, auxin induces ARF production, promoting downstream auxin signal transduction.

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