The Role of Ribosome Biogenesis in Arabidopsis thaliana Root Development and Epidermal Patterning.

The establishment of distinct patterns of gene expression is essential to the differentiation of specialized cell types during the development of multicellular organisms. To investigate the regulatory networks involved in this cell fate specification, one can turn to the Arabidopsis root epidermis, where a position-dependent pattern of hair and non-hair cells serves as a pertinent and accessible model. Recent studies have implicated ribosomal protein and ribosome assembly factors in regulation of gene expression during development, however, the connection between ribosome biogenesis and root cell-type patterning has previously never been investigated. To that end, this thesis research explores the connection and function of a highly conserved ribosome biogenesis factor with respect to the development and epidermal patterning of the Arabidopsis root epidermis. The Arabidopsis DIM1A gene was identified in a genetic screen for genes required for position-dependent expression of non-hair fate regulator GLABRA2. Our analysis has revealed that functional DIM1A is required for generation of distinct gene expression patterns in developing root-hair and non-hair cells. In addition to defects in root epidermal patterning, the dim1A mutant displays reduced root meristem cell division rate and deficient leaf size, shape, vascular patterning and trichome branching. Furthermore, DIM1A promoter activity and gene product are enriched in rapidly dividing plant tissues, supporting a role for DIM1A in Arabidopsis development and cell differentiation. The Dim1/KsgA family of dimethylases are a highly conserved enzymes involved in post-transcriptional modification of ribosomal RNA (rRNA) and pre-rRNA processing. While the two small subunit RNA base modifications they catalyze are present in almost every known organism, the functional significance of these modifications is currently unknown. My experimental evidence indicates that the dim1A mutant lacks the two 18S rRNA modifications without affecting pre-rRNA processing. In conclusion, I propose that DIM1A and the post-transcriptional rRNA modification it catalyzes are important for generating well-defined patterns of gene expression necessary for establishment of the two distinct cell fates in the Arabidopsis root epidermis.Ph.D.Molecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/86319/1/ypancier_1.pd

A Gene Regulatory Network for Root Epidermis Cell Differentiation in Arabidopsis

The root epidermis of Arabidopsis provides an exceptional model for studying the molecular basis of cell fate and differentiation. To obtain a systems-level view of root epidermal cell differentiation, we used a genome-wide transcriptome approach to define and organize a large set of genes into a transcriptional regulatory network. Using cell fate mutants that produce only one of the two epidermal cell types, together with fluorescence-activated cell-sorting to preferentially analyze the root epidermis transcriptome, we identified 1,582 genes differentially expressed in the root-hair or non-hair cell types, including a set of 208 “core” root epidermal genes. The organization of the core genes into a network was accomplished by using 17 distinct root epidermis mutants and 2 hormone treatments to perturb the system and assess the effects on each gene's transcript accumulation. In addition, temporal gene expression information from a developmental time series dataset and predicted gene associations derived from a Bayesian modeling approach were used to aid the positioning of genes within the network. Further, a detailed functional analysis of likely bHLH regulatory genes within the network, including MYC1, bHLH54, bHLH66, and bHLH82, showed that three distinct subfamilies of bHLH proteins participate in root epidermis development in a stage-specific manner. The integration of genetic, genomic, and computational analyses provides a new view of the composition, architecture, and logic of the root epidermal transcriptional network, and it demonstrates the utility of a comprehensive systems approach for dissecting a complex regulatory network

Core root epidermis genes significantly affected in mutant lines.^*

<p>*Significant genes display FC>2 and q<0.05. A list of the gene IDs is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002446#pgen.1002446.s013" target="_blank">Table S5</a>.</p

Three subfamilies of bHLH genes in Arabidopsis involved in root epidermis development.

<p>*The bHLH gene numbering and subfamily organization (based on structural similarities and bHLH domain sequence) have been previously defined <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002446#pgen.1002446-Heim1" target="_blank">[53]</a>.</p><p>**Values represent the average fold-change from a multi-way SAM of <i>wer myb23</i>, <i>ttg</i>, and <i>gl3 egl3</i> versus <i>cpc try</i>. False discovery rate (q-value) is shown in parentheses.</p

Development of the Arabidopsis root epidermis.

<p>Low-magnification images of root tips from Arabidopsis seedling roots showing the series of developmental events that occur from undifferentiated cells (bottom) to mature cells (top). Left: The three major zones of developmental activities are indicated. Scale bar: 100 µm. Right: A root expressing the non-hair cell marker <i>GL2::GUS</i> illustrates the file-specific pattern of developing hair cell files (unstained; indicated as “H”) and non-hair cell files (blue-stained cells). Major epidermal differentiation events are indicated, together with a list of genes known to be involved in each event. Scale bar: 50 µm.</p

Analysis of bHLH transcription factor genes involved in root epidermis development.

<p>(A) Low magnification view of roots from wild-type and homozygous bHLH mutants. Scale bar: 200 µm. (B) High magnification view of individual root hairs from wild-type and each mutant. Scale bar: 30 µm. (C) Cell-type pattern analysis, showing the fraction of root-hair cells and non-hair cells that lie in the H and N cell positions, respectively, of the root epidermis. Mutants which differ significantly from the wild type (p<0.05) are indicated with an asterisk. Some columns lack error bars because all values were identical. (D) Analysis of root hair branching. Mutants which display a significantly greater proportion of branched root hairs than the wild type (p<0.005) are indicated with an asterisk. (E) Expression of the <i>MYC1::GFP</i> transcriptional reporter fusion in the root epidermis of wild-type and mutants. The location of H-cell files is designated by “H”. Scale bar: 20 µm. (F) Root hair length in wild-type and bHLH mutants. The length of full-grown root hairs was measured and the number of hairs in each 50 µm class was determined for each mutant line (white bars) and compared to the wild type (gray bars). Each of the six mutants shown here displayed a significantly different distribution of root hair lengths from wild type (p<0.005). The <i>bHLH69</i> mutant did not exhibit a significant difference in root hair length distribution and is not shown. In panels (C), (D), and (F), error bars indicate standard deviation.</p

Model of the root epidermal gene network.

<p>The predicted transcriptional relationships are shown for the 154 core root hair genes (green), the 54 core non-hair genes (orange), the early acting transcription factors (blue), and other factors not formally part of the network (yellow). The location of genes along the y axis of the figure indicates the relative timing of maximal gene expression during root epidermis development. Genes or gene clusters connected by lines without arrowheads represent genes at a common transcriptional regulatory position but differing in their temporal expression (Zones 1/2, 3/4, and 5/6, from top to bottom). The lists of specific genes in each cluster (A–N) are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002446#pgen.1002446.s016" target="_blank">Table S8</a>, and the GO classes overrepresented in each cluster is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002446#pgen.1002446.s017" target="_blank">Table S9</a>.</p

Effect of mutations on expression of the 208 core root epidermal genes.

<p>(A) Low magnification view of roots from wild-type and homozygous root epidermis mutants. Scale bar: 250 µm. (B) Hierarchical clustering of the 208 core root epidermal genes, based on their relative transcript accumulation in Affymetrix ATH1 microarrays using <i>WER::GFP</i>-expressing cells from three replicates of (left to right) the <i>cpc try</i>, <i>rhd6</i>, <i>gl2</i>, <i>ttg</i>, <i>gl3 egl3</i>, <i>wer myb23</i> mutants and the wild type Columbia. Red = high transcript level; Blue = low transcript level. Asterisks indicate genes significantly affected in the <i>rhd6</i> mutant background. (C) Hierarchical clustering of the 208 core root epidermal genes, based on their relative transcript accumulation (averaged from three replicates) in Affymetrix ATH1 microarrays using <i>WER::GFP</i>-expressing cells from (left to right) <i>cpc try</i>, <i>rhd6</i>, <i>csld3</i>, <i>mrh3</i>, <i>gl2</i>, <i>gl3 egl3</i>, <i>ttg</i>, <i>lrx1</i>, <i>cobl9</i>, <i>rhd2</i>, <i>ire</i>, <i>mrh1</i>, <i>mrh2</i>, <i>wild type</i>, <i>cow1</i>, <i>myc1</i>, <i>bhlh66</i>, and <i>wer myb23</i>. Red = high transcript level; Blue = low transcript level. Heirarchical clustering of the complete set of three replicates for each line is presented as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002446#pgen.1002446.s005" target="_blank">Figure S5</a>.</p