Root Founder Cells Research Articles

CLAVATA3 INSENSITIVE RECEPTOR KINASEs regulate lateral root initiation and spacing in Arabidopsis.

The root system architecture is very critical for plants to adapt to ever-changing environmental stimulations and is largely affected by lateral roots (LRs). Therefore, how plants regulate LR initiation and spacing is a key point for root system development. Previous studies have shown that RECEPTOR-LIKE KINASE 7 (RLK7) and its ligand TARGET OF LBD SIXTEEN 2 (TOLS2) control the initiation and spacing of LRs. However, the molecular mechanism underlying the perception and transduction of the TOLS2 signal by RLK7 remains to be elucidated. In this study, we explored whether CLAVATA3 INSENSITIVE RECEPTOR KINASEs (CIKs) are critical signaling components during Arabidopsis (Arabidopsis thaliana) LR development by investigating phenotypes of cik mutants and examining interactions between CIKs and members of the RLK7-mediated signaling pathway. Our results showed that high-order cik mutants generated more LRs because of more LR initiation and defective LR spacing. The cik mutants showed reduced sensitivity to applied TOLS2 peptides. TOLS2 application enhanced the interactions between CIKs and RLK7 and the RLK7-dependent phosphorylation of CIKs. In addition, overexpression of transcription factor PUCHI and constitutive activation of MITOGEN-ACTIVATED PROTEIN KINASE KINASE 4 (MKK4) and MKK5 partially rescued the spacing defects of LRs in cik and rlk7-3 mutants. Moreover, we discovered that auxin maximum in pericycle cells altered subcellular localization of CIKs to determine lateral root founder cells. These findings revealed that CIKs and RLK7 function together to perceive the TOLS2 signal and regulate LR initiation and spacing through the MKK4/5-MPK3/6-PUCHI cascade.

Trehalose-6-phosphate signaling regulates lateral root formation in Arabidopsis thaliana

Plant roots explore the soil for water and nutrients, thereby determining plant fitness and agricultural yield, as well as determining ground substructure, water levels, and global carbon sequestration. The colonization of the soil requires investment of carbon and energy, but how sugar and energy signaling are integrated with root branching is unknown. Here, we show through combined genetic and chemical modulation of signaling pathways that the sugar small-molecule signal, trehalose-6-phosphate (T6P) regulates root branching through master kinases SNF1-related kinase-1 (SnRK1) and Target of Rapamycin (TOR) and with the involvement of the plant hormone auxin. Increase of T6P levels both via genetic targeting in lateral root (LR) founder cells and through light-activated release of the presignaling T6P-precursor reveals that T6P increases root branching through coordinated inhibition of SnRK1 and activation of TOR. Auxin, the master regulator of LR formation, impacts this T6P function by transcriptionally down-regulating the T6P-degrader trehalose phosphate phosphatase B in LR cells. Our results reveal a regulatory energy-balance network for LR formation that links the 'sugar signal' T6P to both SnRK1 and TOR downstream of auxin.

WOX-ARF modules initiate different types of roots

Seed plants have evolved a complex root system consisting of at least three root types, i.e., adventitious roots, lateral roots, and the primary root. Auxin is the key hormone that controls the initiation of different root types. Here, we show that protein complexes with different combinations of intermediate-clade WUSCHEL-RELATED HOMEOBOXs (IC-WOXs) and class-A AUXIN RESPONSE FACTORs (A-ARFs) initiate the three root types in Arabidopsis thaliana. In adventitious root founder cells from detached leaves, the WOX11-ARF6/8 complex activates RGF1 INSENSITIVEs (RGIs) and LATERAL ORGAN BOUNDARIES DOMAIN 16 (LBD16) to initiate the adventitious root primordium. In lateral root founder cells, ARF7/19 activate RGIs and LBD16 without IC-WOX to initiate the lateral root primordium. In the primary root founder cell (i.e., hypophysis of an embryo), the WOX9-ARF5 complex initiates the primary root by activation of RGIs. Overall, the WOX-ARF modules show a division of labor to initiate different type of roots.

GOLVEN peptides regulate lateral root spacing as part of a negative feedback loop on the establishment of auxin maxima.

Lateral root initiation requires the accumulation of auxin in lateral root founder cells, yielding a local auxin maximum. The positioning of auxin maxima along the primary root determines the density and spacing of lateral roots. The GOLVEN6 (GLV6) and GLV10 signaling peptides and their receptors have been established as regulators of lateral root spacing via their inhibitory effect on lateral root initiation in Arabidopsis. However, it was unclear how these GLV peptides interfere with auxin signaling or homeostasis. Here, we show that GLV6/10 signaling regulates the expression of a subset of auxin response genes, downstream of the canonical auxin signaling pathway, while simultaneously inhibiting the establishment of auxin maxima within xylem-pole pericycle cells that neighbor lateral root initiation sites. We present genetic evidence that this inhibitory effect relies on the activity of the PIN3 and PIN7 auxin export proteins. Furthermore, GLV6/10 peptide signaling was found to enhance PIN7 abundance in the plasma membranes of xylem-pole pericycle cells, which likely stimulates auxin efflux from these cells. Based on these findings, we propose a model in which the GLV6/10 signaling pathway serves as a negative feedback mechanism that contributes to the robust patterning of auxin maxima along the primary root.

Endocytic trafficking induces lateral root founder cell specification in Arabidopsis thaliana in a process distinct from the auxin-induced pathway.

Plants can modify their body structure, such as their root architecture, post-embryonically. For example, Arabidopsis thaliana can develop lateral roots as part of an endogenous program or in response to biotic and abiotic stimuli. Root pericycle cells are specified to become lateral root founder cells, initiating lateral root organogenesis. We used the endocytic trafficking inducer Sortin2 to examine the role of endomembrane trafficking in lateral root founder cell specification. Our results indicate that Sortin2 stimulation turns on a de novo program of lateral root primordium formation that is distinct from the endogenous program driven by auxin. In this distinctive mechanism, extracellular calcium uptake and endocytic trafficking toward the vacuole are required for lateral root founder cell specification upstream of the auxin module led by AUX/IAA28. The auxin-dependent TIR1/AFB F-boxes and auxin polar transport are dispensable for the endocytic trafficking-dependent lateral root founder cell specification; however, a different set of F-box proteins and a functional SCF complex are required. The endocytic trafficking could constitute a convenient strategy for organogenesis in response to environmental conditions.

Microtubule-based perception of mechanical conflicts controls plant organ morphogenesis.

Precise coordination between cells and tissues is essential for differential growth in plants. During lateral root formation in Arabidopsis thaliana, the endodermis is actively remodeled to allow outgrowth of the new organ. Here, we show that microtubule arrays facing lateral root founder cells display a higher order compared to arrays on the opposite side of the same cell, and this asymmetry is required for endodermal remodeling and lateral root initiation. We identify that MICROTUBULE ASSOCIATED PROTEIN 70-5 (MAP70-5) is necessary for the establishment of this spatially defined microtubule organization and endodermis remodeling and thus contributes to lateral root morphogenesis. We propose that MAP70-5 and cortical microtubule arrays in the endodermis integrate the mechanical signals generated by lateral root outgrowth, facilitating the channeling of organogenesis.

Lateral Root Initiation and the Analysis of Gene Function Using Genome Editing with CRISPR in Arabidopsis

Lateral root initiation is a post-embryonic process that requires the specification of a subset of pericycle cells adjacent to the xylem pole in the primary root into lateral root founder cells. The first visible event of lateral root initiation in Arabidopsis is the simultaneous migration of nuclei in neighbouring founder cells. Coinciding cell cycle activation is essential for founder cells in the pericycle to undergo formative divisions, resulting in the development of a lateral root primordium (LRP). The plant signalling molecule, auxin, is a major regulator of lateral root development; the understanding of the molecular mechanisms controlling lateral root initiation has progressed tremendously by the use of the Arabidopsis model and a continual improvement of molecular methodologies. Here, we provide an overview of the visible events, cell cycle regulators, and auxin signalling cascades related to the initiation of a new LRP. Furthermore, we highlight the potential of genome editing technology to analyse gene function in lateral root initiation, which provides an excellent model to answer fundamental developmental questions such as coordinated cell division, growth axis establishment as well as the specification of cell fate and cell polarity.

The TARANI/ UBIQUITIN PROTEASE 14 protein is required for lateral root development in Arabidopsis

ABSTRACT In our article published in Plant Physiology, we had reported tarani (tni) mutant in Arabidopsis, in which poly-ubiquitin hydrolysis is adversely affected, shows pleiotropic phenotypic defects including fewer lateral roots due to the stabilization of several AUX/IAAs and reduced auxin response. TNI encodes UBIQUITIN-SPECIFIC PROTEASE14 that maintains normal auxin response through ubiquitin recycling. Fewer lateral roots observed in tni could be due to defects in their primordia initiation or subsequent elongation post-initiation. Here we have tested this by marking the lateral root primordia with pCycB1;1::CycB1;1(DB):GUS reporter and counting the number of lateral root at various stages development of as a marker of lateral root primordium. The results suggest that TNI/UBP14 is required for LRP development, and a reduction in TNI activity causes a delay in LRP initiation and consequently shorter lateral roots in the tni seedlings. ABBREVIATIONS: LRP, lateral root primordium; XPP, xylem pole pericycle; LRFC, lateral root founder cells

From one cell to many: Morphogenetic field of lateral root founder cells in Arabidopsis thaliana is built by gradual recruitment

The reiterative process of lateral root (LR) formation is widespread and underlies root system formation. However, early LR primordium (LRP) morphogenesis is not fully understood. In this study, we conducted both a clonal analysis and time-lapse experiments to decipher the pattern and sequence of pericycle founder cell (FC) participation in LR formation. Most commonly, LRP initiation starts with the specification of just one FC longitudinally. Clonal and anatomical analyses suggested that a single FC gradually recruits neighboring pericycle cells to become FCs. This conclusion was validated by long-term time-lapse live-imaging experiments. Once the first FC starts to divide, its immediate neighbors, both lengthwise and laterally, are recruited within the hour, after which they recruit their neighboring cells within a few hours. Therefore, LRP initiation is a gradual, multistep process. FC recruitment is auxin-dependent and is abolished by treatment with a polar auxin transport inhibitor. Furthermore, FC recruitment establishes a morphogenetic field where laterally peripheral cells have a lower auxin response, which is associated with a lower proliferation potential, compared to centrally located FCs. The lateral boundaries of the morphogenetic field are determined by phloem-adjacent pericycle cells, which are the last cells to be recruited as FCs. The proliferation potential of these cells is limited, but their recruitment is essential for root system formation, resulting in the formation of a new vascular connection between the nascent and parent root, which is crucial for establishing a continuous and efficient vascular system.

GOLVEN peptide signalling through RGI receptors and MPK6 restricts asymmetric cell division during lateral root initiation.

During lateral root initiation, lateral root founder cells undergo asymmetric cell divisions that generate daughter cells with different sizes and fates, a prerequisite for correct primordium organogenesis. An excess of the GLV6/RGF8 peptide disrupts these initial asymmetric cell divisions, resulting in more symmetric divisions and the failure to achieve lateral root organogenesis. Here, we show that loss-of-function GLV6 and its homologue GLV10 increase asymmetric cell divisions during lateral root initiation, and we identified three members of the RGF1 INSENSITIVE/RGF1 receptor subfamily as likely GLV receptors in this process. Through a suppressor screen, we found that MITOGEN-ACTIVATED PROTEIN KINASE6 is a downstream regulator of the GLV pathway. Our data indicate that GLV6 and GLV10 act as inhibitors of asymmetric cell divisions and signal through RGF1 INSENSITIVE receptors and MITOGEN-ACTIVATED PROTEIN KINASE6 to restrict the number of initial asymmetric cell divisions that take place during lateral root initiation.

Reprogramming of Cell Fate During Root Regeneration by Transcriptional and Epigenetic Networks.

Many plant species are able to regenerate adventitious roots either directly from aerial organs such as leaves or stems, in particularly after detachment (cutting), or indirectly, from over-proliferating tissue termed callus. In agriculture, this capacity of de novo root formation from cuttings can be used to clonally propagate several important crop plants including cassava, potato, sugar cane, banana and various fruit or timber trees. Direct and indirect de novo root regeneration (DNRR) originates from pluripotent cells of the pericycle tissue, from other root-competent cells or from non-root-competent cells that first dedifferentiate. Independently of their origin, the cells convert into root founder cells, which go through proliferation and differentiation subsequently forming functional root meristems, root primordia and the complete root. Recent studies in the model plants Arabidopsis thaliana and rice have identified several key regulators building in response to the phytohormone auxin transcriptional networks that are involved in both callus formation and DNRR. In both cases, epigenetic regulation seems essential for the dynamic reprogramming of cell fate, which is correlated with local and global changes of the chromatin states that might ensure the correct spatiotemporal expression pattern of the key regulators. Future approaches might investigate in greater detail whether and how the transcriptional key regulators and the writers, erasers, and readers of epigenetic modifications interact to control DNRR.

Cytoskeleton Dynamics Are Necessary for Early Events of Lateral Root Initiation in Arabidopsis

How plant cells re-establish differential growth to initiate organs is poorly understood. Morphogenesis of lateral roots relies on the asymmetric cell division of initially symmetric founder cells. This division is preceded by the tightly controlled asymmetric radial expansion of these cells. The cellular mechanisms that license and ensure the coordination of these events are unknown. Here, we quantitatively analyze microtubule and F-actin dynamics during lateral root initiation. Using mutants and pharmacological and tissue-specific genetic perturbations, we show that dynamic reorganization of both microtubule and F-actin networks is necessary for the asymmetric expansion of the founder cells. This cytoskeleton remodeling intertwines with auxin signaling in the pericycle and endodermis in order for founder cells to acquire a basic polarity required for initiating lateral root development. Our results reveal the conservation of cell remodeling and polarization strategies between the Arabidopsis zygote and lateral root founder cells. We propose that coordinated, auxin-driven reorganization of the cytoskeleton licenses asymmetric cell growth and divisions during embryonic and post-embryonic organogenesis.

A Genome-Wide Association Study Identifies New Loci Involved in Wound-Induced Lateral Root Formation in Arabidopsis thaliana.

Root systems can display variable architectures that contribute to nutrient foraging or to increase the tolerance of abiotic stress conditions. Root tip excision promotes the developmental progression of previously specified lateral root (LR) founder cells, which allows to easily measuring the branching capacity of a given root as regards its genotype and/or growth conditions. Here, we describe the natural variation among 120 Arabidopsis thaliana accessions in root system architecture (RSA) after root tip excision. Wound-induced changes in RSA were associated with 19 genomic loci using genome-wide association mapping. Three candidate loci associated with wound-induced LR formation were investigated. Sequence variation in the hypothetical protein encoded by the At4g01090 gene affected wound-induced LR development and its loss-of-function mutants displayed a reduced number of LRs after root tip excision. Changes in a histidine phosphotransfer protein putatively involved in cytokinin signaling were significantly associated with LR number variation after root tip excision. Our results provide a better understanding of some of the genetic components involved in LR capacity variation among accessions.

Long-term live-cell imaging approaches to study lateral root formation in Arabidopsis thaliana.

Lateral roots comprise the majority of the branching root system and are important for acquiring nutrients and water from soil in addition to providing anchorage. Lateral roots develop post-embryonically from existing root parts and originate from a subset of specified pericycle cells (lateral root founder cells) located deep inside roots. Small numbers of these specified pericycle cells undergo several rounds of cell division to create a dome-shaped primordium, which eventually organizes a meristem, an essential region for plant growth with active cell division, and emerges from its parental root as a lateral root. Observing cellular and molecular processes for an extended time at various scales are crucial for understanding biological processes during organogenesis. Lateral root formation is an example of the successful application of live-cell imaging approaches to understand various aspects of developmental events in plants, including cell fate determination, cell proliferation, cell-to-cell interaction and cell wall modification. Here I review the recent progress in understanding the molecular mechanisms of lateral root formation and the contribution of live-cell imaging approaches.

Recruitment of IC-WOX Genes in Root Evolution.

Root evolution has resulted in the extant bifurcating roots in lycophytes, adventitious/lateral roots in euphyllophytes (ferns and seed plants), and primary roots in seed plants. Here, we hypothesize a role for intermediate-clade-WUSCHEL-RELATED HOMEOBOX (IC-WOX) genes in root evolution. IC-WOX might not be specifically involved in lycophyte bifurcation rooting. In the fern Ceratopteris richardii, IC-WOX is expressed in adventitious/lateral root founder cells. In the seed plant Arabidopsis thaliana, there are two IC-WOX subclades, AtWOX11/12 and AtWOX8/9, in adventitious and primary root founder cells, respectively. Thus, IC-WOX was recruited in the common ancestor of ferns and seed plants for adventitious/lateral root organogenesis and evolved into two subclades in seed plants: one was retained in adventitious root organogenesis, while the other was recruited for primary root organogenesis.

Regulation of Hormonal Control, Cell Reprogramming, and Patterning during De Novo Root Organogenesis

Body regeneration through formation of new organs is a major question in developmental biology. We investigated de novo root formation using whole leaves of Arabidopsis (Arabidopsis thaliana). Our results show that local cytokinin biosynthesis and auxin biosynthesis in the leaf blade followed by auxin long-distance transport to the petiole leads to proliferation of J0121-marked xylem-associated tissues and others through signaling of INDOLE-3-ACETIC ACID INDUCIBLE28 (IAA28), CRANE (IAA18), WOODEN LEG, and ARABIDOPSIS RESPONSE REGULATORS1 (ARR1), ARR10, and ARR12. Vasculature proliferation also involves the cell cycle regulator KIP-RELATED PROTEIN2 and ABERRANT LATERAL ROOT FORMATION4, resulting in a mass of cells with rooting competence that resembles callus formation. Endogenous callus formation precedes specification of postembryonic root founder cells, from which roots are initiated through the activity of SHORT-ROOT, PLETHORA1 (PLT1), and PLT2. Primordia initiation is blocked in shr plt1 plt2 mutant. Stem cell regulators SCHIZORIZA, JACKDAW, BLUEJAY, and SCARECROW also participate in root initiation and are required to pattern the new organ, as mutants show disorganized and reduced number of layers and tissue initials resulting in reduced rooting. Our work provides an organ regeneration model through de novo root formation, stating key stages and the primary pathways involved.

Lateral root formation and the multiple roles of auxin.

Root systems can display variable architectures that contribute to survival strategies of plants. The model plant Arabidopsis thaliana possesses a tap root system, in which the primary root and lateral roots (LRs) are major architectural determinants. The phytohormone auxin fulfils multiple roles throughout LR development. In this review, we summarize recent advances in our understanding of four aspects of LR formation: (i) LR positioning, which determines the spatial distribution of lateral root primordia (LRP) and LRs along primary roots; (ii) LR initiation, encompassing the activation of nuclear migration in specified lateral root founder cells (LRFCs) up to the first asymmetric cell division; (iii) LR outgrowth, the 'primordium-intrinsic' patterning of de novo organ tissues and a meristem; and (iv) LR emergence, an interaction between LRP and overlaying tissues to allow passage through cell layers. We discuss how auxin signaling, embedded in a changing developmental context, plays important roles in all four phases. In addition, we discuss how rapid progress in gene network identification and analysis, modeling, and four-dimensional imaging techniques have led to an increasingly detailed understanding of the dynamic regulatory networks that control LR development.

Transcription Factors WOX11/12 Directly Activate WOX5/7 to Promote Root Primordia Initiation and Organogenesis.

De novo organogenesis, which gives rise to adventitious roots and shoots, is a type of plant regeneration for survival after wounding. In Arabidopsis (Arabidopsis thaliana), two main cell fate transition steps are required to establish the root primordium during de novo root organogenesis from leaf explants. The first step from regeneration-competent cells to root founder cells involves activation of WUSCHEL-RELATED HOMEOBOX11 (WOX11) and WOX12 (WOX11/12) expression by auxin. However, the molecular mechanism controlling the second step of fate transition from root founder cells to root primordium is poorly understood. In this study, we show that the expression levels of WOX11/12 decrease while those of WOX5 and 7 (WOX5/7) increase during the transition from root founder cells to the root primordium. WOX11/12 function genetically upstream of WOX5/7, and the WOX11/12 proteins directly bind to the promoters of WOX5/7 to activate their transcription. Mutations in WOX5/7 result in defective primordium formation. Overall, our data indicate that the expression switch from WOX11/12 to WOX5/7 is critical for initiation of the root primordium during de novo root organogenesis.

YUCCA-mediated auxin biogenesis is required for cell fate transition occurring during de novo root organogenesis in Arabidopsis.

Many plant organs have the ability to regenerate a new plant after detachment or wounding via de novo organogenesis. During de novo root organogenesis from Arabidopsis thaliana leaf explants, endogenic auxin is essential for the fate transition of regeneration-competent cells to become root founder cells via activation of WUSCHEL-RELATED HOMEOBOX 11 (WOX11). However, the molecular events from leaf explant detachment to auxin-mediated cell fate transition are poorly understood. In this study, we used an assay to determine the concentration of indole-3-acetic acid (IAA) to provide direct evidence that auxin is produced after leaf explant detachment, a process that involves YUCCA (YUC)-mediated auxin biogenesis. Inhibition of YUC prevents expression of WOX11 and fate transition of competent cells, resulting in the blocking of rooting. Further analysis showed that YUC1 and YUC4 act quickly (within 4 hours) in response to wounding after detachment in both light and dark conditions and promote auxin biogenesis in both mesophyll and competent cells, whereas YUC5, YUC8, and YUC9 primarily respond in dark conditions. In addition, YUC2 and YUC6 contribute to rooting by providing a basal auxin level in the leaf. Overall, our study indicates that YUC genes exhibit a division of labour during de novo root organogenesis from leaf explants in response to multiple signals.

The cyclophilin A DIAGEOTROPICA gene affects auxin transport in both root and shoot to control lateral root formation.

Cyclophilin A is a conserved peptidyl-prolyl cis-trans isomerase (PPIase) best known as the cellular receptor of the immunosuppressant cyclosporine A. Despite significant effort, evidence of developmental functions of cyclophilin A in non-plant systems has remained obscure. Mutations in a tomato (Solanum lycopersicum) cyclophilin A ortholog, DIAGEOTROPICA (DGT), have been shown to abolish the organogenesis of lateral roots; however, a mechanistic explanation of the phenotype is lacking. Here, we show that the dgt mutant lacks auxin maxima relevant to priming and specification of lateral root founder cells. DGT is expressed in shoot and root, and localizes to both the nucleus and cytoplasm during lateral root organogenesis. Mutation of ENTIRE/IAA9, a member of the auxin-responsive Aux/IAA protein family of transcriptional repressors, partially restores the inability of dgt to initiate lateral root primordia but not the primordia outgrowth. By comparison, grafting of a wild-type scion restores the process of lateral root formation, consistent with participation of a mobile signal. Antibodies do not detect movement of the DGT protein into the dgt rootstock; however, experiments with radiolabeled auxin and an auxin-specific microelectrode demonstrate abnormal auxin fluxes. Functional studies of DGT in heterologous yeast and tobacco-leaf auxin-transport systems demonstrate that DGT negatively regulates PIN-FORMED (PIN) auxin efflux transporters by affecting their plasma membrane localization. Studies in tomato support complex effects of the dgt mutation on PIN expression level, expression domain and plasma membrane localization. Our data demonstrate that DGT regulates auxin transport in lateral root formation.