Browsing by Author "Benfey, Philip N"

The Arabidopsis root apical meristem (RAM) is a complex tissue capable of generating all the cell types that ultimately make up the root. The work presented in this thesis takes advantage of the versatility of high-throughput sequencing to address two independent questions about the root meristem. Although a lot of information is known regarding the cell fate decisions that occur at the RAM, cortex specification and differentiation remain poorly understood. In the first part of this thesis, I used an ethylmethanesulfonate (EMS) mutagenized marker line to perform a forward genetics screen. The goal of this screen was to identify novel genes involved in the specification and differentiation of the cortex tissue. Mapping analysis from the results obtained in this screen revealed a new allele of BRASSINOSTEROID4 with abnormal marker expression in the cortex tissue. Although this allele proved to be non-cortex specific, this project highlights new technology that allows mapping of EMS-generated mutations without the need to map-cross or back-cross. In the second part of this thesis, using fluorescence activated cell sorting (FACS) coupled with high throughput sequencing, my collaborators and I generated single-base resolution whole genome DNA methylomes, mRNA transcriptomes, and smallRNA transcriptomes for six different populations of cell types in the Arabidopsis root meristem. We were able to discover that the columella is hypermethylated in the CHH context within transposable elements. This hypermethylation is accompanied by upregulation of the RNA-dependent DNA methylation pathway (RdDM), including higher levels of 24-nt silencing RNAs (siRNAs). In summary, our studies demonstrate the versatility of high-throughput sequencing as a method for identifying single mutations or to perform complex comparative genomic analyses.

This research spans multiple scales—from the entire organism, down to the genes that created it.The first project, “Gene Dynamics in Tissue Development”, explores how stem cell differentiation depends on the dynamics of gene networks. In the Arabidopsis thaliana root, the SCARECROW (SCR) transcription factor is required for an asymmetric cell division of a stem cell, resulting in two daughter cells that acquire different fates and tissue identities. Although much research has developed the network topology for this division, the dynamics of this process remain unknown. A core feature of the GRN controlling this stem cell asymmetric division is the SCR positive feedback loop. This research develops a synthetic biology approach to systematically and precisely tune various dynamics of SCR protein accumulation. Thus, one can explore the role and function of this positive feedback loop in the developmental process of asymmetric division in the Arabidopsis root. The following project, “Organ Form for Function” details how organ function depends on cellular form and hormonal signals. As sessile organisms, plants must establish a firm foundation into the terrain wherever the seed lands. Roots, especially the primary root (a seed’s first root), are the only anchor into the terrain. With a multiscale investigation, we identified a molecular pathway required for circumnutation, the circular growth of the root tip. We found the cellular physiology and key hormonal cell signaling events driving this behavior.

Plants have evolved a robust immune system to fend off pathogens. This response must be tightly regulated, as aberrant activation can have detrimental effects. Much work has been done to understand the transcriptional responses in plant immunity, but less is known about post-transcriptional mechanisms. Here I examine the roles of the general control nonderepressible 2 (GCN2) kinase and the N6-methyladenosine (m6A) RNA modification in regulating plant immunity at the post-transcriptional level.

A previous study revealed that the master immune regulator TBF1 (a transcription factor) is essential for mediating a defense response. TBF1 is under the control of 2 upstream open reading frames (uORFs) which inhibit translation of TBF1’s major open reading frame (mORF). These uORFs’ sequences are enriched with codons for aromatic amino acids, especially phenylalanine. Pathogen treatment resulted in the induction of uncharged tRNAphe and eIF2α phosphorylation. It was proposed that GCN2 would mediate eIF2α phosphorylation and allow readthrough of TBF1’s mORF, analogous to GCN2’s role in promoting translation of GCN4 upon amino acid starvation. To test this, eIF2α phosphorylation was examined in gcn2 mutant plants upon pathogen infection using a phoshpo-specific eIF2α antibody. eIF2α phosphorylation was not observed in gcn2 plants upon pathogen treatment and therefore GCN2 is likely to be the kinase responsible for this induced eIF2α phosphorylation. To test GCN2’s role in positively regulating TBF1, a bacterial infection assay was undertaken with gcn2 plants. Compared to wild-type plants, gcn2 plants did not have any significant difference in bacterial growth and therefore GCN2 is unlikely to play a role in regulating TBF1 or plant immunity.

The m6A modification is the most abundant internal modification present in mRNAs and has been found to regulate several aspects of mRNA metabolism and biological processes. There is less known about m6A in plants, though it has been found to regulate stability of transcripts and is important for development and the salt stress response. It is unknown whether m6A plays a role in plant immunity. To address this, m6A deficient plant lines were used and bacterial infection assays undertaken. These lines displayed significantly higher levels of bacterial growth (susceptibility) and thus m6A plays a positive role in plant immunity. Further assays found that m6A was essential for fully mediating pattern-triggered immunity (PTI) and salicylic acid (SA)-mediated immune responses. m6A-seq was used to map the dynamics of m6A across the transcriptome in response to the immune inducers SA and elf18 (a microbe-associated molecular pattern). Hundreds of pathogen-induced methylation sites were uncovered and gene ontology (GO) analysis revealed a predominance of defense/immune-related transcripts.

In summary, this dissertation work found that although GCN2 is required for pathogen induced eIF2α phosphorylation, it is dispensable for defense against a bacterial pathogen, and thus is unlikely to be a regulator of TBF1 or the immune response. On the other hand, m6A was established to be broadly essential and dynamic upon immune induction. These findings open the door for future studies to elucidate how m6A machinery is interacting with the defense response and establish a new area for post-transcriptional control of plant immunity.

An uncertain climate, paired with rapid human population growth, presents a major challenge to maintaining food security in the twenty-first century. Improvement of cultivation of rice, a primary source of calories for nearly half of the world’s people, provides a unique opportunity to address this challenge. As breeding in cereals has largely focused on aboveground phenotypes, root system traits represent potential unexplored targets for stress resilience and yield improvement. However, our understanding of the genetic control of root system architecture (RSA) in rice is fundamentally insufficient to contribute to these goals. The identification of novel rice RSA loci and the genes that underlie them could potentially provide breeders the tools to test the effect of root traits on water and nutrient usage.

Our lab has developed a gel-based imaging and phenotyping system to facilitate genetic mapping of root traits in rice. Using this, we identify a mutation in a gene encoding a putative rice histidine kinase (OsHK1) that results in plants with increased seedling root depth. Using time-lapse imaging, we show that OsHK1 mutants have reduced circumnutation, or circular root tip growth. This supports a previously underappreciated link between RSA and the underlying pattern of root growth.

Environmental stress affects plant development and productivity. Sulfur deficiency is a key nutrient deficiency that adversely affects crop yield. The model plant Arabidopsis thaliana has played an informative role in deciphering the mechanisms involved in sulfur assimilation, as well as, the response to limited conditions. Using Arabidopsis thaliana as a model to investigate gene expression in the root, microarray data sets have been generated. These data sets consist of whole root sections for 6 time points across 72 hours, and enriched populations of 5 radial cell-types and 4 sections of 3 developmental zones of the root at 3 hrs on sulfur limited conditions. With these data it was determined which cellular tissues and developmental zones were affected most by sulfur limited conditions. Furthermore, a novel phenotype was characterized that occurs in roots after growth on low sulfur conditions. Cellular inclusions build up within the cytoplasm of mature cortical root cells. These inclusions have been termed "sulfur pox" and their composition remains to be determined.

When a plant emerges from the soil, it faces a critical developmental transition from utilizing stored energy to grow rapidly toward the light, to developing chloroplasts and beginning photosynthesis. While it is known that this process involves massive transcriptional reprogramming of the nuclear and plastidial genomes, the connections between chloroplast development and nuclear light signaling events are not well understood. One very promising target for investigating these connections is HEMERA (HMR), a dual-localized regulatory protein that is found in both nuclei and chloroplasts. HMR was previously identified as pTAC12, an essential component of the plastid-encoded RNA polymerase complex responsible for transcription of chloroplast photosynthetic genes. In the nucleus, HMR acts within the phytochrome signaling pathway as a transcriptional co-activator of a subset of growth-relevant genes in response to light, to regulate the elongation of the embryonic stem, or hypocotyl. HMR’s combination of roles in the nucleus and chloroplasts are dramatically demonstrated by the phenotypes of the hmr mutant, with a long hypocotyl and albino leaves when grown in the light.

While the functions of HMR in each compartment have been studied separately, the mechanisms by which the HMR protein is targeted to each compartment have not yet been determined. To address this, I characterized the localization signals of HMR with a combination of in vitro approaches and characterization of transgenic Arabidopsis lines. These experiments revealed that HMR has a cleavable N-terminal chloroplast transit peptide within its first 50 amino acids, while two predicted nuclear localization signals proved not to be highly functional. Surprisingly, HMR in the chloroplasts and nucleus appeared to both be the same cleaved form of the protein. We thus identified the mature form of HMR by mass spectrometry, finding that it begins from lysine as the result of transit peptide cleavage and possibly additional N-terminal processing. Through GST pull-down assays, we determined that this mature form of HMR was fully capable of interacting light signaling components. However, analysis of transgenic lines showed that expression of mature HMR alone could not complement the long-hypocotyl phenotype of the hmr mutant. Analysis of the transcription of HMR nuclear target genes confirmed that mature HMR lacked nuclear functionality.

Further investigation revealed that mature HMR does not accumulate within the nucleus, most likely as a result of its nonfunctional nuclear localization signals. However, addition of the transit peptide from the small subunit of Rubisco fully restored nuclear accumulation and function of mature HMR in Arabidopsis. Additional experiments testing the localization of a simple model of dual-targeted proteins with two types of localization signal showed that transit peptides might take priority over nuclear localization signals. These results together suggest an unexpected model of localization where HMR is first targeted to the chloroplasts, and then it is subsequently re-localized to the nucleus, thus connecting its nuclear and plastidial functions. Further investigation of this proposed retrograde plastid-to-nucleus translocation pathway promises to shed additional light on the link between nuclear light signaling events and chloroplast development.

A cell’s trajectory from stem cell to differentiation, while often portrayed as a linear progression, is best described as a network that produces a mature state through several pathways acting together. There are few examples that describe gene regulatory network changes during the entire trajectory of cell differentiation. The goal of my project was to define the gene regulatory network required for a stem cell to become a differentiated cell in the Arabidopsis thaliana root. The root is a powerful model for identifying basic principles of differentiation. Plant cells do not migrate therefore entire lineages from stem cell to mature progeny are spatially confined. Furthermore, the root displays indeterminate growth, facilitating the study of many different developmental stages at a single time. One cell type of the root, the endodermis, is particularly suitable for study because the molecular components required for its formation and terminal differentiation are established. In order to understand the path from stem cell to differentiated cell in the endodermis, we asked what transcription factors are sufficient to program a non-native cell-type into endodermis. Our results show the transcription factors SHORTROOT and MYB36 have limited ability to reprogram a non-native cell-type (the epidermis) and that this reprogramming is reversible in the absence of additional cues. The stele-derived signaling peptide CIF2 stabilizes SHORTROOT-induced reprogramming. The outcome is a partially impermeable barrier deposited in the sub-epidermal cell layer that has a transcriptional signature similar to endodermis. The induction mechanism depends on MYB36 and CIF2’s receptor, but may be independent of the transcription factor SCARECROW. These results highlight a non cell-autonomous induction mechanism for endodermis that resembles differentiation in many animal systems.

Root system architecture (RSA) is the spatial distribution of roots of individual plants. As part of a collaborative effort I adapted a gellan gum based system for imaging and phenotyping of root systems in maize. This system was first used to perform a survey of 26 distinct maize varieties of the Nested Association Mapping (NAM) population. The analysis of these data showed a large amount of variation between different RSA, in particular demonstrating tradeoffs between architectures favoring sparse, but far reaching, root networks versus those favoring small but dense root networks. To study this further I imaged and phenotyped the B73 (compact) x Ki3 (exploratory) mapping population. These data were used to map 102 quantitative trait loci (QTL). A large portion of these QTL had large, ranging from 5.48% to 23.8%. Majority of these QTLs were grouped into 9 clusters across the genome, with each cluster favoring either the compact of exploratory RSA. In summary, our study demonstrates the power of the gellan based system to locate loci controlling root system architecture of maize, by combining rapid and highly detailed imaging techniques with semi-automated computation phenotyping.

The successful completion of many critical biological processes depends on the proper execution of complex spatial and temporal gene expression programs. With the advent of high-throughput microarray technology, it is now possible to measure the dynamics of these expression programs on a genome-wide level. In this thesis we present work focused on utilizing this technology, in combination with novel computational techniques, to examine the role of transcriptional regulatory mechanisms in controlling the complex gene expression programs underlying two fundamental biological processes---the cell cycle and the development and differentiation of an organ.

We generate a dataset describing the genomic expression program which occurs during the cell division cycle of Saccharomyces cerevisiae. By concurrently measuring the dynamics in both wild-type and mutant cells that do not express either S-phase or mitotic cyclins we quantify the relative contributions of cyclin-CDK complexes and transcriptional regulatory networks in the regulation the cell cell expression program. We show that CDKs are not the sole regulators of periodic transcription as contrary to previously accepted models; and we hypothesize an oscillating transcriptional regulatory network which could work independent of, or in tandem with, the CDK oscillator to control the cell cell expression program.

To understand the acquisition of cellular identity, we generate a nearly complete gene expression map of the Arabidopsis Thaliana root at the resolution of individual cell-types and developmental stages. An analysis of this data reveals a representative set of dominant expression patterns which are used to begin defining the spatiotemporal transcriptional programs that control development within the root.

Additionally, we develop computational tools that improve the interpretability and power of these data. We present CLOCCS, a model for the dynamics of population synchrony loss in time-series experiments. We demonstrate the utility of CLOCCS in integrating disparate datasets and present a CLOCCS based deconvolution of the cell-cycle expression data. A deconvolution method is also developed for the Arabidopsis dataset, increasing its resolution to cell-type/section subregion specificity. Finally, a method for identifying biological processes occurring on multiple timescales is presented and applied to both datasets.

It is through the combination of these new genome-wide expression studies and computational tools that we begin to elucidate the transcriptional regulatory mechanisms controlling fundamental biological processes.

Small noncoding RNAs (ncRNAs) are key regulators of plant development through modulation of the processing, stability and translation of larger RNAs. I generated small RNA datasets comprising over 200 million aligned Illumina sequence reads covering all major cell types of the root as well as four distinct developmental zones. These data were analyzed for three major types of small RNAs, namely microRNAs (miRNAs), repeat associated small interfering RNAs (ra-siRNAs), and trans-acting siRNAs (ta-siRNAs). 133 of the 243 known miRNAs were found to be expressed in the root, and most showed tissue- or zone-specific expression patterns. My collaborators and I identified 70 new high-confidence miRNAs, and knockdown of three of the newly identified miRNAs resulted in altered root growth phenotypes. Ra-siRNAs specify methylation by the RNA directed DNA methylation (RdRM) pathway, requiring the generation of additional methylation datasets. Preliminary analysis shows cell-type specific methylation patterns that correlate with small RNA and mRNA expression. Analysis of ta-siRNAs revealed new ta-siRNA generating loci, and a novel triggering miRNA for TAS1 loci. In summary, our study demonstrates the power of isolating individual cell types and developmental zones in combination with deep sequencing and computational analyses to obtain detailed profiles of ncRNAs, as well as to significantly extend the compendium of known functional RNAs.

Roots are developmentally plastic and highly dependent on the immediate environment. By studying root responses to abiotic stress, we have identified novel regulators of developmental modulation. When roots are deprived of phosphate (Pi), developmental programs are modulated to slow primary root growth and expand surface area through emergence of root hairs. By focusing on exposure time-periods of less than two days, we have described very early changes to root development in response to this condition that may reveal new mechanisms of root hair specification and emergence. Also, using transcriptomic analyses with high spatial resolution, we identified a kinase that is specifically induced in root vascular tissue within three hours of exposure and acts to modulate aspects of root development in response to deprivation of Pi. These data suggest that individual tissues play unique roles in whole organ development, and that interpretation of Pi -deprivation responses may change as we develop methods with resolution necessary to understand these roles. Beyond Pi, we compared transcriptomic data for four additional stresses and identified a novel stress-responsive transcription factor that modulates expression of a cell expansion protein. This putative network connection demonstrates the value of using high-dimensional data for inference of regulatory relationships. Overall, we have combined "-omics" approaches with reverse genetics to identify novel developmental regulators and described a phenotypic frame-work with resolution at which cellular mechanisms can be studied.