Project description:The goal of this experiment was to identify the downstream targets of the GOLVEN6 peptide signaling pathway in Arabidopsis thaliana, specifically during lateral root initiation. Using an estradiol inducible GLV6 overexpression construct in wildtype and rgi1rgi5 double mutant (mutant in receptors for the GLV6 peptide) backgrounds, in combination with gravistimulation induced lateral root formation, the RGI receptor dependent transcriptional effects of GLV6 overexpression were characterized. An estradiol inducible GLV6 overexpression line in a wildtype (iGLV6) and in an rgi1rgi5 double receptor mutant background (rgi1rgi5/iGLV6) were used. 4-day old seedlings of both lines were gravistimulated (vertically grown seedlings were turned counterclockwise by 90°) to induce lateral root initiation in the resulting root bends. 8h after gravistimulation, seedlings of both lines were treated with 2µM of estradiol to induce GLV6 overexpression, or DMSO as a mock treatment. 3h and 6h after treatment, root bends were dissected and collected for RNA-sequencing. This yielded a total of 8 samples per replicate; 3h mock treated iGLV6 (IM3), 3h estradiol treated iGLV6 (IE3), 3h mock treated rgi1rgi5/iGLV6 (RM3), 3h estradiol treated rgi1rgi5/iGLV6 (RE3), 6h mock treated iGLV6 (IM6), 6h estradiol treated iGLV6 (IE6), 6h mock treated rgi1rgi5/iGLV6 (RM6), 6h estradiol treated rgi1rgi5/iGLV6 (RE6). For each sample, 4 replicates were obtained. This setup enabled the comparison of the GLV6 induced transcriptional effects between wildtype and rgi1rgi5 mutants at 2 time points after treatment, in samples that are strongly enriched for lateral root initiation events.
Project description:Plant root architecture is a major determinant of fitness, and is under constant modification in response to favorable and unfavorable environmental stimuli. Beyond impacts on the primary root, the environment can also alter the position, spacing, density, and length of secondary or lateral roots. Lateral root development is among the best-studied developmental processes in Arabidopsis thaliana, yet the earliest steps of organogenesis remain elusive. Among the challenges faced in capturing these early molecular events is the fact that this process occurs in a small number of cells with unpredictable timing. The advent of single-cell sequencing affords the opportunity to isolate cells undergoing this fate transition and examine their transcriptomes independently. Using this approach, we successfully captured the transcriptomes of lateral root primordia and discovered many previously unreported upregulated genes. To further study this process, we developed a method to selectively repress genes in the xylem pole pericycle cells where lateral roots originate. We found that expression of several of the upregulated genes was required for normal root development. In addition, we discovered a subpopulation of cells in the endodermal cell file that respond to lateral root initiation, further highlighting the benefits of the single cell approach.
Project description:Control of the dimensions of organ primordia is crucial for proper organogenesis in the development of multicellular organisms. Lateral root formation is a major type of plant organogenesis important for postembryonic development of the root system. Lateral root formation begins with a few rounds of asymmetric, anticlinal cell division (formative cell division) in the pericycle, which determines the basal dimensions of root primordia. Here we show, based on molecular genetic analysis of temperature-dependent fasciation (TDF) mutants of Arabidopsis thaliana, that mitochondria play an unexpected role in the restriction of formative cell division and thus in the control of the basal dimensions of lateral root primordia. Three TDF mutants, root redifferentiation defective 1 (rrd1), rrd2, and root initiation defective 4 (rid4), exhibit lateral root fasciation from excess formative cell division under high-temperature conditions. We identify RRD1 as encoding a poly(A)-specific ribonuclease (PARN)-like protein and RRD2 and RID4 as encoding pentatricopeptide repeat (PPR) proteins. Subcellular localization and predicted functions of these proteins implicate them in poly(A)-dependent RNA degradation in mitochondria. This characterization is supported by the finding that mitochondrial RNAs with poly(A) tails, most of which are mRNAs of respiratory chain components, accumulate at an unusually high level in these TDF mutants.
Project description:The acquisition of water and nutrients by plant roots is a fundamental aspect of agriculture and strongly depends on root architecture. Root branching and expansion of the root system is achieved through the development of lateral roots and is to a large extent controlled by the plant hormone auxin. However, the pleiotropic effects of auxin or auxin-like molecules on root systems complicate the study of lateral root development. Here we describe a small-molecule screen in Arabidopsis thaliana that identified naxillin as what is to our knowledge the first non-auxin-like molecule that promotes root branching. By using naxillin as a chemical tool, we identified a new function for root cap-specific conversion of the auxin precursor indole-3-butyric acid into the active auxin indole-3-acetic acid and uncovered the involvement of the root cap in root branching. Delivery of an auxin precursor in peripheral tissues such as the root cap might represent an important mechanism shaping root architecture. To further explore the specificity of naxillin for lateral root development, we compared the early effects of naxillin at the transcriptome level with NAA (1-Naphthaleneacetic acid) in roots of 3-day-old seedlings after 2-h and 6-h treatment.
Project description:The acquisition of water and nutrients by plant roots is a fundamental aspect of agriculture and strongly depends on root architecture. Root branching and expansion of the root system is achieved through the development of lateral roots and is to a large extent controlled by the plant hormone auxin. However, the pleiotropic effects of auxin or auxin-like molecules on root systems complicate the study of lateral root development. Here we describe a small-molecule screen in Arabidopsis thaliana that identified naxillin as what is to our knowledge the first non-auxin-like molecule that promotes root branching. By using naxillin as a chemical tool, we identified a new function for root cap-specific conversion of the auxin precursor indole-3-butyric acid into the active auxin indole-3-acetic acid and uncovered the involvement of the root cap in root branching. Delivery of an auxin precursor in peripheral tissues such as the root cap might represent an important mechanism shaping root architecture. To further explore the specificity of naxillin for lateral root development, we compared the early effects of naxillin at the transcriptome level with NAA (1-Naphthaleneacetic acid) in roots of 3-day-old seedlings after 2-h and 6-h treatment. Arabidopsis thaliana (L). Heynh., Col-0 seeds were germinated vertically on solid medium derived from standard MS medium supplemented with 10 μM NPA (1-N-Naphthylphthalamic acid). Three days after germination, plants were transferred to 10 μM NAA (1-Naphthaleneacetic acid) or 50 μM naxillin for 2 and 6 hours. Plants were sampled before (Roots at T0, NPA) or after treatment (Roots at T1 and T2). RNA isolation was performed on 500 root sections (only root without meristems) for each sample. All sampling points were performed in three independent experiments.
Project description:Lateral root initiation was used as a model system to study the mechanisms behind auxin-induced cell division. 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 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. Experiment Overall Design: Seedlings of both wild type (Col-0) and the lateral root defective mutant (slr-1) were germinated on MS medium supplemented with 10μM NPA (=auxin transport inhibitor). Three days after germination, such seedlings were transferred to MS supplemented with 10μM NAA for 0h, 2h and 6h respectively. The segment between root meristem and root-hypocotyl junction was harvested from about 1500 seedling per time point. All treatments were repeated biologically. 5.8 μg total RNA was used for the preparation of biotinylated cRNA. Labeled RNA was hybridised to ATH1 Affymetrix chips. The resulting data was MAS5.0 normalised.
Project description:Control of the dimensions of organ primordia is crucial for proper organogenesis in the development of multicellular organisms. Lateral root formation is a major type of plant organogenesis important for postembryonic development of the root system. Lateral root formation begins with a few rounds of asymmetric, anticlinal cell division (formative cell division) in the pericycle, which determines the basal dimensions of root primordia. Here we show, based on molecular genetic analysis of temperature-dependent fasciation (TDF) mutants of Arabidopsis thaliana, that mitochondria play an unexpected role in the restriction of formative cell division and thus in the control of the basal dimensions of lateral root primordia. Three TDF mutants, root redifferentiation defective 1 (rrd1), rrd2, and root initiation defective 4 (rid4), exhibit lateral root fasciation from excess formative cell division under high-temperature conditions. We identify RRD1 as encoding a poly(A)-specific ribonuclease (PARN)-like protein and RRD2 and RID4 as encoding pentatricopeptide repeat (PPR) proteins. Subcellular localization and predicted functions of these proteins implicate them in poly(A)-dependent RNA degradation in mitochondria. This characterization is supported by the finding that mitochondrial RNAs with poly(A) tails, most of which are mRNAs of respiratory chain components, accumulate at an unusually high level in these TDF mutants. Explants of three mutants (rrd1, rrd2, rid4) and wild type were cultured on B5 medium supplemented with 0.5 mg/L IBA for the induction of lateral root formation. After 12 hours of culture at 28C, explants were collected and used for microarray analysis. Analysis was performed in three biological replicates.
Project description:This model is from the article:
The influence of cytokinin-auxin cross-regulation on cell-fate determination in Arabidopsis thaliana root development
Muraro D, Byrne H, King J, Voss U, Kieber J, Bennett M.
J Theor Biol.2011 Aug 21;283(1):152-67.
PMID: 21640126,
Abstract:
Root growth and development in Arabidopsis thaliana are sustained by a specialised zone termed the meristem, which contains a population of dividing and differentiating cells that are functionally analogous to a stem cell niche in animals. The hormones auxin and cytokinin control meristem size antagonistically. Local accumulation of auxin promotes cell division and the initiation of a lateral root primordium. By contrast, high cytokinin concentrations disrupt the regular pattern of divisions that characterises lateral root development, and promote differentiation. The way in which the hormones interact is controlled by a genetic regulatory network. In this paper, we propose a deterministic mathematical model to describe this network and present model simulations that reproduce the experimentally observed effects of cytokinin on the expression of auxin regulated genes. We show how auxin response genes and auxin efflux transporters may be affected by the presence of cytokinin. We also analyse and compare the responses of the hormones auxin and cytokinin to changes in their supply with the responses obtained by genetic mutations of SHY2, which encodes a protein that plays a key role in balancing cytokinin and auxin regulation of meristem size. We show that although shy2 mutations can qualitatively reproduce the effect of varying auxin and cytokinin supply on their response genes, some elements of the network respond differently to changes in hormonal supply and to genetic mutations, implying a different, general response of the network. We conclude that an analysis based on the ratio between these two hormones may be misleading and that a mathematical model can serve as a useful tool for stimulate further experimental work by predicting the response of the network to changes in hormone levels and to other genetic mutations.
Project description:The RETINOBLASTOMA–RELATED (RBR) is a key regulator of cell proliferation and differentiation in plants, and plays an important role in maintenance of the stem cell niche in the root. We used microarray analysis to characterize the transcriptional response of Arabidopsis thaliana root tips from rRBr mutant (7 samples) against Col-0 wild type (6 samples) after 4, 6 and 10 das.