Small-molecule screens to study lateral root development

Development of the root system is essential for proper plant growth and development. Extension of the root system is achieved by the continuous establishment of new meristems in existing parental root tissues, which leads to the development of lateral roots. This process of lateral root organogenesis consists of different developmental stages, which are all controlled by the plant hormone auxin. In this chapter, we describe a screening method in Arabidopsis thaliana to identify small synthetic molecules that interfere with the process of lateral root development during specific developmental stages

An Auxin Transport-Based Model of Root Branching in Arabidopsis thaliana

Root architecture is a crucial part of plant adaptation to soil heterogeneity and is mainly controlled by root branching. The process of root system development can be divided into two successive steps: lateral root initiation and lateral root development/emergence which are controlled by different fluxes of the plant hormone auxin. While shoot architecture appears to be highly regular, following rules such as the phyllotactic spiral, root architecture appears more chaotic. We used stochastic modeling to extract hidden rules regulating root branching in Arabidopsis thaliana. These rules were used to build an integrative mechanistic model of root ramification based on auxin. This model was experimentally tested using plants with modified rhythm of lateral root initiation or mutants perturbed in auxin transport. Our analysis revealed that lateral root initiation and lateral root development/emergence are interacting with each other to create a global balance between the respective ratio of initiation and emergence. A mechanistic model based on auxin fluxes successfully predicted this property and the phenotype alteration of auxin transport mutants or plants with modified rythms of lateral root initiation. This suggests that root branching is controlled by mechanisms of lateral inhibition due to a competition between initiation and development/emergence for auxin

Sequential induction of auxin efflux and influx carriers regulates lateral root emergence

Peer reviewe

An ancient and conserved function for armadillo-related proteins in the control of spore and seed germination by abscisic acid

Armadillo‐related proteins regulate development throughout eukaryotic kingdoms. In the flowering plant Arabidopsis thaliana, Armadillo‐related ARABIDILLO proteins promote multicellular root branching. ARABIDILLO homologues exist throughout land plants, including early‐diverging species lacking true roots, suggesting that early‐evolving ARABIDILLOs had additional biological roles. Here we investigated, using molecular genetics, the conservation and diversification of ARABIDILLO protein function in plants separated by c. 450 million years of evolution. We demonstrate that ARABIDILLO homologues in the moss Physcomitrella patens regulate a previously undiscovered inhibitory effect of abscisic acid (ABA) on spore germination. Furthermore, we show that A. thaliana ARABIDILLOs function similarly during seed germination. Early‐diverging ARABIDILLO homologues from both P. patens and the lycophyte Selaginella moellendorffii can substitute for ARABIDILLO function during A. thaliana root development and seed germination. We conclude that (1) ABA was co‐opted early in plant evolution to regulate functionally analogous processes in spore‐ and seed‐producing plants and (2) plant ARABIDILLO germination functions were co‐opted early into both gametophyte and sporophyte, with a specific rooting function evolving later in the land plant lineage

Quantitation of Cellular Dynamics in Growing Arabidopsis Roots with Light Sheet Microscopy

To understand dynamic developmental processes, living tissues must be imaged frequently and for extended periods of time. Root development is extensively studied at cellular resolution to understand basic mechanisms underlying pattern formation and maintenance in plants. Unfortunately, ensuring continuous specimen access, while preserving physiological conditions and preventing photo-damage, poses major barriers to measurements of cellular dynamics in indeterminately growing organs such as plant roots. We present a system that integrates optical sectioning through light sheet fluorescence microscopy with hydroponic culture that enables us to image at cellular resolution a vertically growing Arabidopsis root every few minutes and for several consecutive days. We describe novel automated routines to track the root tip as it grows, track cellular nuclei and identify cell divisions. We demonstrate the system's capabilities by collecting data on divisions and nuclear dynamics.Comment: * The first two authors contributed equally to this wor

A Systems Approach Uncovers Restrictions for Signal Interactions Regulating Genome-wide Responses to Nutritional Cues in Arabidopsis

As sessile organisms, plants must cope with multiple and combined variations of signals in their environment. However, very few reports have studied the genome-wide effects of systematic signal combinations on gene expression. Here, we evaluate a high level of signal integration, by modeling genome-wide expression patterns under a factorial combination of carbon (C), light (L), and nitrogen (N) as binary factors in two organs (O), roots and leaves. Signal management is different between C, N, and L and in shoots and roots. For example, L is the major factor controlling gene expression in leaves. However, in roots there is no obvious prominent signal, and signal interaction is stronger. The major signal interaction events detected genome wide in Arabidopsis roots are deciphered and summarized in a comprehensive conceptual model. Surprisingly, global analysis of gene expression in response to C, N, L, and O revealed that the number of genes controlled by a signal is proportional to the magnitude of the gene expression changes elicited by the signal. These results uncovered a strong constraining structure in plant cell signaling pathways, which prompted us to propose the existence of a “code” of signal integration

A Novel fry1 Allele Reveals the Existence of a Mutant Phenotype Unrelated to 5′->3′ Exoribonuclease (XRN) Activities in Arabidopsis thaliana Roots

International audienceBackgroundMutations in the FRY1/SAL1 Arabidopsis locus are highly pleiotropic, affecting drought tolerance, leaf shape and root growth. FRY1 encodes a nucleotide phosphatase that in vitro has inositol polyphosphate 1-phosphatase and 3′,(2′),5′-bisphosphate nucleotide phosphatase activities. It is not clear which activity mediates each of the diverse biological functions of FRY1 in planta.Principal FindingsA fry1 mutant was identified in a genetic screen for Arabidopsis mutants deregulated in the expression of Pi High affinity Transporter 1;4 (PHT1;4). Histological analysis revealed that, in roots, FRY1 expression was restricted to the stele and meristems. The fry1 mutant displayed an altered root architecture phenotype and an increased drought tolerance. All of the phenotypes analyzed were complemented with the AHL gene encoding a protein that converts 3′-polyadenosine 5′-phosphate (PAP) into AMP and Pi. PAP is known to inhibit exoribonucleases (XRN) in vitro. Accordingly, an xrn triple mutant with mutations in all three XRNs shared the fry1 drought tolerance and root architecture phenotypes. Interestingly these two traits were also complemented by grafting, revealing that drought tolerance was primarily conferred by the rosette and that the root architecture can be complemented by long-distance regulation derived from leaves. By contrast, PHT1 expression was not altered in xrn mutants or in grafting experiments. Thus, PHT1 up-regulation probably resulted from a local depletion of Pi in the fry1 stele. This hypothesis is supported by the identification of other genes modulated by Pi deficiency in the stele, which are found induced in a fry1 background.Conclusions/SignificanceOur results indicate that the 3′,(2′),5′-bisphosphate nucleotide phosphatase activity of FRY1 is involved in long-distance as well as local regulatory activities in roots. The local up-regulation of PHT1 genes transcription in roots likely results from local depletion of Pi and is independent of the XRNs.

The AUXIN BINDING PROTEIN 1 Is Required for Differential Auxin Responses Mediating Root Growth

Background In plants, the phytohormone auxin is a crucial regulator sustaining growth and development. At the cellular level, auxin is interpreted differentially in a tissue- and dose-dependent manner. Mechanisms of auxin signalling are partially unknown and the contribution of the AUXIN BINDING PROTEIN 1 (ABP1) as an auxin receptor is still a matter of debate. Methodology/Principal Findings Here we took advantage of the present knowledge of the root biological system to demonstrate that ABP1 is required for auxin response. The use of conditional ABP1 defective plants reveals that the protein is essential for maintenance of the root meristem and acts at least on the D-type CYCLIN/RETINOBLASTOMA pathway to control entry into the cell cycle. ABP1 affects PLETHORA gradients and confers auxin sensitivity to root cells thus defining the competence of the cells to be maintained within the meristem or to elongate. ABP1 is also implicated in the regulation of gene expression in response to auxin. Conclusions/Significance Our data support that ABP1 is a key regulator for root growth and is required for auxin-mediated responses. Differential effects of ABP1 on various auxin responses support a model in which ABP1 is the major regulator for auxin action on the cell cycle and regulates auxin-mediated gene expression and cell elongation in addition to the already well known TIR1-mediated ubiquitination pathway

The OSU1/QUA2/TSD2-Encoded Putative Methyltransferase Is a Critical Modulator of Carbon and Nitrogen Nutrient Balance Response in Arabidopsis

The balance between carbon (C) and nitrogen (N) nutrients must be tightly coordinated so that cells can optimize their opportunity for metabolism, growth and development. However, the C and N nutrient balance perception and signaling mechanism remains poorly understood. Here, we report the isolation and characterization of two allelic oversensitive to sugar1 mutants (osu1-1, osu1-2) in Arabidopsis thaliana. Using the cotyledon anthocyanin accumulation and root growth inhibition assays, we show that the osu1 mutants are more sensitive than wild-type to both of the imbalanced C/N conditions, high C/low N and low C/high N. However, under the balanced C/N conditions (low C/low N or high C/high N), the osu1 mutants have similar anthocyanin levels and root lengths as wild-type. Consistently, the genes encoding two MYB transcription factors (MYB75 and MYB90) and an Asn synthetase isoform (ASN1) are strongly up-regulated by the OSU1 mutation in response to high C/low N and low C/high N, respectively. Furthermore, the enhanced sensitivity of osu1-1 to high C/low N with respect to anthocyanin accumulation but not root growth inhibition can be suppressed by co-suppression of MYB75, indicating that MYB75 acts downstream of OSU1 in the high C/low N imbalance response. Map-based cloning reveals that OSU1 encodes a member of a large family of putative methyltransferases and is allelic to the recently reported QUA2/TSD2 locus identified in genetic screens for cell-adhesion-defective mutants. Accumulation of OSU1/QUA2/TSD2 transcript was not regulated by C and N balance, but the OSU1 promoter was slightly more active in the vascular system. Taken together, our results show that the OSU1/QUA2/TSD2-encoded putative methyltransferase is required for normal C/N nutrient balance response in plants