Interspecies co-expression analysis of lateral root development using inducible systems in rice, Medicago, and Arabidopsis.

Lateral roots are crucial for plant growth and development, making them an important target for research aiming to improve crop yields and food security. However, their endogenous ontogeny and, as it were, stochastic appearance challenge their study. Lateral Root Inducible Systems (LRIS) can be used to overcome these challenges by inducing lateral roots massively and synchronously. The combination of LRISs with transcriptomic approaches significantly advanced our insights in the molecular control of lateral root formation, in particular for Arabidopsis. Despite this success, LRISs have been underutilized for other plant species or for lateral root developmental stages later than the initiation. In this study, we developed and/or adapted LRISs in rice, Medicago, and Arabidopsis to perform RNA-sequencing during time courses that cover different developmental stages of lateral root formation and primordium development. As such, our study provides three extensive datasets of gene expression profiles during lateral root development in three different plant species. The three LRISs are highly effective but timing and spatial distribution of lateral root induction vary among the species. Detailed characterization of the stages in time and space in the respective species enabled an interspecies co-expression analysis to identify conserved players involved in lateral root development, as illustrated for the AUX/IAA and LBD gene families. Overall, our results provide a valuable resource to identify potentially conserved regulatory mechanisms in lateral root development, and as such will contribute to a better understanding of the complex regulatory network underlying lateral root development.

Translational profile of developing phellem cells in Arabidopsis thaliana roots.

The phellem is a specialized boundary tissue providing the first line of defense against abiotic and biotic stresses in organs undergoing secondary growth. Phellem cells undergo several differentiation steps, which include cell wall suberization, cell expansion, and programmed cell death. Yet, the molecular players acting particularly in phellem cell differentiation remain poorly described, particularly in the widely used model plant Arabidopsis thaliana. Using specific marker lines we followed the onset and progression of phellem differentiation in A. thaliana roots and further targeted the translatome of newly developed phellem cells using translating ribosome affinity purification followed by mRNA sequencing (TRAP-SEQ). We showed that phellem suberization is initiated early after phellogen (cork cambium) division. The specific translational landscape was organized in three main domains related to energy production, synthesis and transport of cell wall components, and response to stimulus. Novel players in phellem differentiation related to suberin monomer transport and assembly as well as novel transcription regulators were identified. This strategy provided an unprecedented resolution of the translatome of developing phellem cells, giving a detailed and specific view on the molecular mechanisms acting on cell differentiation in periderm tissues of the model plant Arabidopsis.

CROWN ROOTLESS1 binds DNA with a relaxed specificity and activates OsROP and OsbHLH044 genes involved in crown root formation in rice.

In cereals, the root system is mainly composed of post-embryonic shoot-borne roots, named crown roots. The CROWN ROOTLESS1 (CRL1) transcription factor, belonging to the ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES DOMAIN (ASL/LBD) family, is a key regulator of crown root initiation in rice (Oryza sativa). Here, we show that CRL1 can bind, both in vitro and in vivo, not only the LBD-box, a DNA sequence recognized by several ASL/LBD transcription factors, but also another not previously identified DNA motif that was named CRL1-box. Using rice protoplast transient transactivation assays and a set of previously identified CRL1-regulated genes, we confirm that CRL1 transactivates these genes if they possess at least a CRL1-box or an LBD-box in their promoters. In planta, ChIP-qPCR experiments targeting two of these genes that include both a CRL1- and an LBD-box in their promoter show that CRL1 binds preferentially to the LBD-box in these promoter contexts. CRISPR/Cas9-targeted mutation of these two CRL1-regulated genes, which encode a plant Rho GTPase (OsROP) and a basic helix-loop-helix transcription factor (OsbHLH044), show that both promote crown root development. Finally, we show that OsbHLH044 represses a regulatory module, uncovering how CRL1 regulates specific processes during crown root formation.

Cellular and gene expression patterns associated with root bifurcation in Selaginella.

The roots of lycophytes branch through dichotomy or bifurcation, during which the root apex splits into two daughter roots. This is morphologically distinct from lateral root (LR) branching in the extant euphyllophytes, with LRs developing along the root axis at different distances from the apex. Although the process of root bifurcation is poorly understood, such knowledge can be important, because it may represent an evolutionarily ancient strategy that roots recruited to form new stem cells or meristems. In this study, we examined root bifurcation in the lycophyte Selaginella moellendorffii. We characterized an in vitro developmental time frame based on repetitive apex bifurcations, allowing us to sample different stages of dichotomous root branching and analyze the root meristem and root branching in S. moellendorffii at the microscopic and transcriptomic level. Our results showed that, in contrast to previous assumptions, initial cells (ICs) in the root meristem are mostly not tetrahedral but rather show an irregular shape. Tracking down the early stages of root branching argues for the occurrence of a symmetric division of the single IC, resulting in two apical stem cells that initiate root meristem bifurcation. Moreover, we generated a S. moellendorffii root branching transcriptome that resulted in the delineation of a subset of core meristem genes. The occurrence of multiple putative orthologs of meristem genes in this dataset suggests the presence of conserved pathways in the control of meristem and root stem cell establishment or maintenance.

Two phylogenetically unrelated peptide-receptor modules jointly regulate lateral root initiation via a partially shared signaling pathway in Arabidopsis thaliana.

Peptide-receptor signaling is an important system for intercellular communication, regulating many developmental processes. A single process can be controlled by several distinct signaling peptides. However, since peptide-receptor modules are usually studied separately, their mechanistic interactions remain largely unexplored. Two phylogenetically unrelated peptide-receptor modules, GLV6/GLV10-RGI and TOLS2/PIP2-RLK7, independently described as inhibitors of lateral root initiation, show striking similarities between their expression patterns and gain- and loss-of-function phenotypes, suggesting a common function during lateral root spacing and initiation. The GLV6/GLV10-RGI and TOLS2/PIP2-RLK7 modules trigger similar transcriptional changes, likely in part via WRKY transcription factors. Their overlapping set of response genes includes PUCHI and PLT5, both required for the effect of GLV6/10, as well as TOLS2, on lateral root initiation. Furthermore, both modules require the activity of MPK6 and can independently trigger MPK3/MPK6 phosphorylation. The GLV6/10 and TOLS2/PIP2 signaling pathways seem to converge in the activation of MPK3/MPK6, leading to the induction of a similar transcriptional response in the same target cells, thereby regulating lateral root initiation through a (partially) common mechanism. Convergence of signaling pathways downstream of phylogenetically unrelated peptide-receptor modules adds an additional, and hitherto unrecognized, level of complexity to intercellular communication networks in plants.

Rice plants respond to ammonium stress by adopting a helical root growth pattern.

High levels of ammonium nutrition reduce plant growth and different plant species have developed distinct strategies to maximize ammonium acquisition while alleviating ammonium toxicity through modulating root growth. To date, the mechanisms underlying plant tolerance or sensitivity towards ammonium remain unclear. Rice (Oryza sativa) uses ammonium as its main N source. Here we show that ammonium supply restricts rice root elongation and induces a helical growth pattern, which is attributed to root acidification resulting from ammonium uptake. Ammonium-induced low pH triggers the asymmetric distribution of auxin in rice root tips through changes in auxin signaling, thereby inducing a helical growth response. Blocking auxin signaling completely inhibited this root response. In contrast, this root response is not activated in ammonium-treated Arabidopsis. Acidification of Arabidopsis roots leads to the protonation of indole-3-acetic acid and dampening of the intracellular auxin signaling levels that are required for maintaining root growth. Our study suggests a different mode of action by ammonium on the root pattern and auxin response machinery in rice versus Arabidopsis, and the rice-specific helical root response towards ammonium is an expression of the ability of rice to moderate auxin signaling and root growth to utilize ammonium while confronting acidic stress.

NAC Transcription Factors ANAC087 and ANAC046 Control Distinct Aspects of Programmed Cell Death in the Arabidopsis Columella and Lateral Root Cap.

Programmed cell death in plants occurs both during stress responses and as an integral part of regular plant development. Despite the undisputed importance of developmentally controlled cell death processes for plant growth and reproduction, we are only beginning to understand the underlying molecular genetic regulation. Exploiting the Arabidopsis thaliana root cap as a cell death model system, we identified two NAC transcription factors, the little-characterized ANAC087 and the leaf-senescence regulator ANAC046, as being sufficient to activate the expression of cell death-associated genes and to induce ectopic programmed cell death. In the root cap, these transcription factors are involved in the regulation of distinct aspects of programmed cell death. ANAC087 orchestrates postmortem chromatin degradation in the lateral root cap via the nuclease BFN1. In addition, both ANAC087 and ANAC046 redundantly control the onset of cell death execution in the columella root cap during and after its shedding from the root tip. Besides identifying two regulators of developmental programmed cell death, our analyses reveal the existence of an actively controlled cell death program in Arabidopsis columella root cap cells.

Exploiting natural variation in root system architecture via genome-wide association studies.

Root growth and development has become an important research topic for breeders and researchers based on a growing need to adapt plants to changing and more demanding environmental conditions worldwide. Over the last few years, genome-wide association studies (GWASs) became an important tool to identify the link between traits in the field and their genetic background. Here we give an overview of the current literature concerning GWASs performed on root system architecture (RSA) in plants. We summarize which root traits and approaches have been used for GWAS, mentioning their respective success rate towards a successful gene discovery. Furthermore, we zoom in on the current technical hurdles in root phenotyping and GWAS, and discuss future possibilities in this field of research.

RBOH-mediated ROS production facilitates lateral root emergence in Arabidopsis.

Lateral root (LR) emergence represents a highly coordinated process in which the plant hormone auxin plays a central role. Reactive oxygen species (ROS) have been proposed to function as important signals during auxin-regulated LR formation; however, their mode of action is poorly understood. Here, we report that Arabidopsis roots exposed to ROS show increased LR numbers due to the activation of LR pre-branch sites and LR primordia (LRP). Strikingly, ROS treatment can also restore LR formation in pCASP1:shy2-2 and aux1 lax3 mutant lines in which auxin-mediated cell wall accommodation and remodeling in cells overlying the sites of LR formation is disrupted. Specifically, ROS are deposited in the apoplast of these cells during LR emergence, following a spatiotemporal pattern that overlaps the combined expression domains of extracellular ROS donors of the RESPIRATORY BURST OXIDASE HOMOLOGS (RBOH). We also show that disrupting (or enhancing) expression of RBOH in LRP and/or overlying root tissues decelerates (or accelerates) the development and emergence of LRs. We conclude that RBOH-mediated ROS production facilitates LR outgrowth by promoting cell wall remodeling of overlying parental tissues.

OsMADS26 Negatively Regulates Resistance to Pathogens and Drought Tolerance in Rice.

Functional analyses of MADS-box transcription factors in plants have unraveled their role in major developmental programs (e.g. flowering and floral organ identity) as well as stress-related developmental processes, such as abscission, fruit ripening, and senescence. Overexpression of the rice (Oryza sativa) MADS26 gene in rice has revealed a possible function related to stress response. Here, we show that OsMADS26-down-regulated plants exhibit enhanced resistance against two major rice pathogens: Magnaporthe oryzae and Xanthomonas oryzae. Despite this enhanced resistance to biotic stresses, OsMADS26-down-regulated plants also displayed enhanced tolerance to water deficit. These phenotypes were observed in both controlled and field conditions. Interestingly, alteration of OsMADS26 expression does not have a strong impact on plant development. Gene expression profiling revealed that a majority of genes misregulated in overexpresser and down-regulated OsMADS26 lines compared with control plants are associated to biotic or abiotic stress response. Altogether, our data indicate that OsMADS26 acts as an upstream regulator of stress-associated genes and thereby, a hub to modulate the response to various stresses in the rice plant.

Expanding the repertoire of secretory peptides controlling root development with comparative genome analysis and functional assays.

Plant genomes encode numerous small secretory peptides (SSPs) whose functions have yet to be explored. Based on structural features that characterize SSP families known to take part in postembryonic development, this comparative genome analysis resulted in the identification of genes coding for oligopeptides potentially involved in cell-to-cell communication. Because genome annotation based on short sequence homology is difficult, the criteria for the de novo identification and aggregation of conserved SSP sequences were first benchmarked across five reference plant species. The resulting gene families were then extended to 32 genome sequences, including major crops. The global phylogenetic pattern common to the functionally characterized SSP families suggests that their apparition and expansion coincide with that of the land plants. The SSP families can be searched online for members, sequences and consensus (http://bioinformatics.psb.ugent.be/webtools/PlantSSP/). Looking for putative regulators of root development, Arabidopsis thaliana SSP genes were further selected through transcriptome meta-analysis based on their expression at specific stages and in specific cell types in the course of the lateral root formation. As an additional indication that formerly uncharacterized SSPs may control development, this study showed that root growth and branching were altered by the application of synthetic peptides matching conserved SSP motifs, sometimes in very specific ways. The strategy used in the study, combining comparative genomics, transcriptome meta-analysis and peptide functional assays in planta, pinpoints factors potentially involved in non-cell-autonomous regulatory mechanisms. A similar approach can be implemented in different species for the study of a wide range of developmental programmes.

Analyzing lateral root development: how to move forward.

Roots are important to plants for a wide variety of processes, including nutrient and water uptake, anchoring and mechanical support, storage functions, and as the major interface between the plant and various biotic and abiotic factors in the soil environment. Therefore, understanding the development and architecture of roots holds potential for the manipulation of root traits to improve the productivity and sustainability of agricultural systems and to better understand and manage natural ecosystems. While lateral root development is a traceable process along the primary root and different stages can be found along this longitudinal axis of time and development, root system architecture is complex and difficult to quantify. Here, we comment on assays to describe lateral root phenotypes and propose ways to move forward regarding the description of root system architecture, also considering crops and the environment.

Identification of potential transcriptional regulators of actinorhizal symbioses in Casuarina glauca and Alnus glutinosa.

BACKGROUND: Trees belonging to the Casuarinaceae and Betulaceae families play an important ecological role and are useful tools in forestry for degraded land rehabilitation and reforestation. These functions are linked to their capacity to establish symbiotic relationships with a nitrogen-fixing soil bacterium of the genus Frankia. However, the molecular mechanisms controlling the establishment of these symbioses are poorly understood. The aim of this work was to identify potential transcription factors involved in the establishment and functioning of actinorhizal symbioses. RESULTS: We identified 202 putative transcription factors by in silico analysis in 40 families in Casuarina glauca (Casuarinaceae) and 195 in 35 families in Alnus glutinosa (Betulaceae) EST databases. Based on published transcriptome datasets and quantitative PCR analysis, we found that 39% and 26% of these transcription factors were regulated during C. glauca and A. glutinosa-Frankia interactions, respectively. Phylogenetic studies confirmed the presence of common key transcription factors such as NSP, NF-YA and ERN-related proteins involved in nodule formation in legumes, which confirm the existence of a common symbiosis signaling pathway in nitrogen-fixing root nodule symbioses. We also identified an actinorhizal-specific transcription factor belonging to the zinc finger C1-2i subfamily we named CgZF1 in C. glauca and AgZF1 in A. glutinosa. CONCLUSIONS: We identified putative nodulation-associated transcription factors with particular emphasis on members of the GRAS, NF-YA, ERF and C2H2 families. Interestingly, comparison of the non-legume and legume TF with signaling elements from actinorhizal species revealed a new subgroup of nodule-specific C2H2 TF that could be specifically involved in actinorhizal symbioses. In silico identification, transcript analysis, and phylogeny reconstruction of transcription factor families paves the way for the study of specific molecular regulation of symbiosis in response to Frankia infection.

Auxin-dependent cell cycle reactivation through transcriptional regulation of Arabidopsis E2Fa by lateral organ boundary proteins.

Multicellular organisms depend on cell production, cell fate specification, and correct patterning to shape their adult body. In plants, auxin plays a prominent role in the timely coordination of these different cellular processes. A well-studied example is lateral root initiation, in which auxin triggers founder cell specification and cell cycle activation of xylem pole-positioned pericycle cells. Here, we report that the E2Fa transcription factor of Arabidopsis thaliana is an essential component that regulates the asymmetric cell division marking lateral root initiation. Moreover, we demonstrate that E2Fa expression is regulated by the LATERAL ORGAN BOUNDARY DOMAIN18/LATERAL ORGAN BOUNDARY DOMAIN33 (LBD18/LBD33) dimer that is, in turn, regulated by the auxin signaling pathway. LBD18/LBD33 mediates lateral root organogenesis through E2Fa transcriptional activation, whereas E2Fa expression under control of the LBD18 promoter eliminates the need for LBD18. Besides lateral root initiation, vascular patterning is disrupted in E2Fa knockout plants, similarly as it is affected in auxin signaling and lbd mutants, indicating that the transcriptional induction of E2Fa through LBDs represents a general mechanism for auxin-dependent cell cycle activation. Our data illustrate how a conserved mechanism driving cell cycle entry has been adapted evolutionarily to connect auxin signaling with control of processes determining plant architecture.

A role for the root cap in root branching revealed by the non-auxin probe naxillin.

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.

Comparative transcriptomics as a tool for the identification of root branching genes in maize.

The root system is fundamental for plant development, is crucial for overall plant growth and is recently being recognized as the key for future crop productivity improvement. A major determinant of root system architecture is the initiation of lateral roots. While knowledge of the genetic and molecular mechanisms regulating lateral root initiation has mainly been achieved in the dicotyledonous plant Arabidopsis thaliana, only scarce data are available for major crop species, generally monocotyledonous plants. The existence of both similarities and differences at the morphological and anatomical level between plant species from both clades raises the question whether regulation of lateral root initiation may or may not be conserved through evolution. Here, we performed a targeted genome-wide transcriptome analysis during lateral root initiation both in primary and in adventitious roots of Zea mays and found evidence for the existence of common transcriptional regulation. Further, based on a comparative analysis with Arabidopsis transcriptome data, a core of genes putatively conserved across angiosperms could be identified. Therefore, it is plausible that common regulatory mechanisms for lateral root initiation are at play in maize and Arabidopsis, a finding that might encourage the extrapolation of knowledge obtained in Arabidopsis to crop species at the level of root system architecture.

VisuaLRTC: a new view on lateral root initiation by combining specific transcriptome data sets.

Lateral root initiation and development has been increasingly studied over the last two decades. This postembryonic organogenic process guarantees the spatial development and plasticity of the root system in response to environmental cues and is crucial for the plant's growth and development. Several independent large-scale transcriptome studies in different species resulted in a wealth of data that can be instructive to understand this process at the molecular level. Here, we present an easy and flexible spreadsheet tool, called Visual Lateral Root Transcriptome Compendium, that combines publicly available data sets involved in Arabidopsis (Arabidopsis thaliana) lateral root development and links them with additional information on tissue-specific expression and cell cycle involvement, thus allowing the extraction of novel information from existing data sets in a visual and user-friendly manner. We believe that this tool will be valuable not only for root biologists but also for a broader range of scientists as it enables a fast indication of the potential involvement of a given gene during de novo organogenesis.

Early "Rootprints" of Plant Terrestrialization: Selaginella Root Development Sheds Light on Root Evolution in Vascular Plants.

Roots provide multiple key functions for plants, including anchorage and capturing of water and nutrients. Evolutionarily, roots represent a crucial innovation that enabled plants to migrate from aquatic to terrestrial environment and to grow in height. Based on fossil evidence, roots evolved at least twice independently, once in the lycophyte clade and once in the euphyllophyte (ferns and seed plants) clade. In lycophytes, roots originated in a stepwise manner. Despite their pivotal position in root evolution, it remains unclear how root development is controlled in lycophytes. Getting more insight into lycophyte root development might shed light on how genetic players controlling the root meristem and root developmental processes have evolved. Unfortunately, genetic studies in lycophytes are lagging behind, lacking advanced biotechnological tools, partially caused by the limited economic value of this clade. The technology of RNA sequencing (RNA-seq) at least enabled transcriptome studies, which could enhance the understanding or discovery of genes involved in the root development of this sister group of euphyllophytes. Here, we provide an overview of the current knowledge on root evolution followed by a survey of root developmental events and how these are genetically and hormonally controlled, starting from insights obtained in the model seed plant Arabidopsis and where possible making a comparison with lycophyte root development. Second, we suggest possible key genetic regulators in root development of lycophytes mainly based on their expression profiles in Selaginella moellendorffii and phylogenetics. Finally, we point out challenges and possible future directions for research on root evolution.

Genetic Variability of Arabidopsis thaliana Mature Root System Architecture and Genome-Wide Association Study.

Root system architecture (RSA) has a direct influence on the efficiency of nutrient uptake and plant growth, but the genetics of RSA are often studied only at the seedling stage. To get an insight into the genetic blueprint of a more mature RSA, we exploited natural variation and performed a detailed in vitro study of 241 Arabidopsis thaliana accessions using large petri dishes. A comprehensive analysis of 17 RSA traits showed high variability among the different accessions, unveiling correlations between traits and conditions of the natural habitat of the plants. A sub-selection of these accessions was grown in water-limiting conditions in a rhizotron set-up, which revealed that especially the spatial distribution showed a high consistency between in vitro and ex vitro conditions, while in particular, a large root area in the lower zone favored drought tolerance. The collected RSA phenotype data were used to perform genome-wide association studies (GWAS), which stands out from the previous studies by its exhaustive measurements of RSA traits on more mature Arabidopsis accessions used for GWAS. As a result, we found not only several genes involved in the lateral root (LR) development or auxin signaling pathways to be associated with RSA traits but also new candidate genes that are potentially involved in the adaptation to the natural habitats.

Periodic root branching is influenced by light through an HY1-HY5-auxin pathway.

The spacing of lateral roots (LRs) along the main root in plants is driven by an oscillatory signal, often referred to as the "root clock" that represents a pre-patterning mechanism that can be influenced by environmental signals. Light is an important environmental factor that has been previously reported to be capable of modulating the root clock, although the effect of light signaling on the LR pre-patterning has not yet been fully investigated. In this study, we reveal that light can activate the transcription of a photomorphogenic gene HY1 to maintain high frequency and amplitude of the oscillation signal, leading to the repetitive formation of pre-branch sites. By grafting and tissue-specific complementation experiments, we demonstrated that HY1 generated in the shoot or locally in xylem pole pericycle cells was sufficient to regulate LR branching. We further found that HY1 can induce the expression of HY5 and its homolog HYH, and act as a signalosome to modulate the intracellular localization and expression of auxin transporters, in turn promoting auxin accumulation in the oscillation zone to stimulate LR branching. These fundamental mechanistic insights improve our understanding of the molecular basis of light-controlled LR formation and provide a genetic interconnection between shoot- and root-derived signals in regulating periodic LR branching.