The Root Cap Cuticle: A Cell Wall Structure for Seedling Establishment and Lateral Root Formation.

The root cap surrounding the tip of plant roots is thought to protect the delicate stem cells in the root meristem. We discovered that the first layer of root cap cells is covered by an electron-opaque cell wall modification resembling a plant cuticle. Cuticles are polyester-based protective structures considered exclusive to aerial plant organs. Mutations in cutin biosynthesis genes affect the composition and ultrastructure of this cuticular structure, confirming its cutin-like characteristics. Strikingly, targeted degradation of the root cap cuticle causes a hypersensitivity to abiotic stresses during seedling establishment. Furthermore, lateral root primordia also display a cuticle that, when defective, causes delayed outgrowth and organ deformations, suggesting that it facilitates lateral root emergence. Our results show that the previously unrecognized root cap cuticle protects the root meristem during the critical phase of seedling establishment and promotes the efficient formation of lateral roots.

Autophagy promotes organelle clearance and organized cell separation of living root cap cells in Arabidopsis thaliana.

The root cap is a multilayered tissue covering the tip of a plant root that directs root growth through its unique functions, such as gravity sensing and rhizosphere interaction. To maintain the structure and function of the root cap, its constituent cells are constantly turned over through balanced cell division and cell detachment in the inner and outer cell layers, respectively. Upon displacement toward the outermost layer, columella cells at the central root cap domain functionally transition from gravity-sensing cells to secretory cells, but the mechanisms underlying this drastic cell fate transition are largely unknown. Here, using live-cell tracking microscopy, we show that organelles in the outermost cell layer undergo dramatic rearrangements. This rearrangement depends, at least partially, on spatiotemporally regulated activation of autophagy. Notably, this root cap autophagy does not lead to immediate cell death, but is instead necessary for organized separation of living root cap cells, highlighting a previously undescribed role of developmentally regulated autophagy in plants. This article has an associated 'The people behind the papers' interview.

Early sensing of phosphate deprivation triggers the formation of extra root cap cell layers via SOMBRERO through a process antagonized by auxin signaling.

KEY MESSAGE: The role of the root cap in the plant response to phosphate deprivation has been scarcely investigated. Here we describe early structural, physiological and molecular changes prior to the determinate growth program of the primary roots under low Pi and unveil a critical function of the transcription factor SOMBRERO in low Pi sensing. Mineral nutrient distribution in the soil is uneven and roots efficiently adapt to improve uptake and assimilation of sparingly available resources. Phosphate (Pi) accumulates in the upper layers and thus short and branched root systems proliferate to better exploit organic and inorganic Pi patches. Here we report an early adaptive response of the Arabidopsis primary root that precedes the entrance of the meristem into the determinate developmental program that is a hallmark of the low Pi sensing mechanism. In wild-type seedlings transferred to low Pi medium, the quiescent center domain in primary root tips increases as an early response, as revealed by WOX5:GFP expression and this correlates with a thicker root tip with extra root cap cell layers. The halted primary root growth in WT seedlings could be reversed upon transfer to medium supplemented with 250 µM Pi. Mutant and gene expression analysis indicates that auxin signaling negatively affects the cellular re-specification at the root tip and enabled identification of the transcription factor SOMBRERO as a critical element that orchestrates both the formation of extra root cap layers and primary root growth under Pi scarcity. Moreover, we provide evidence that low Pi-induced root thickening or the loss-of-function of SOMBRERO is associated with expression of phosphate transporters at the root tip. Our data uncover a developmental window where the root tip senses deprivation of a critical macronutrient to improve adaptation and surveillance.

CRISPR-TSKO: A Technique for Efficient Mutagenesis in Specific Cell Types, Tissues, or Organs in Arabidopsis.

Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (Arabidopsis thaliana), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of system-wide losses of gene function.

Root cap size and shape influence responses to the physical strength of the growth medium in Arabidopsis thaliana primary roots.

During the progression of root in soil, root cap cells are the first to encounter obstacles and are known to sense environmental cues, thus making the root cap a potential mechanosensing site. In this study, a two-layered growth medium system was developed in order to study root responses to variations in the physical strength of the medium and the importance of the root cap in the establishment of these responses. Root growth and trajectory of primary roots of Arabidopsis seedlings were investigated using in vivo image analysis. After contact with the harder layer of the medium, the root either penetrated it or underwent rapid curvature, thus enabling reorientation of growth. We initially hypothesized that the root-cap structure would affect apex penetration and reorientation, with pointed caps facilitating and domed caps impeding root penetration. This hypothesis was investigated by analysing the responses of Arabidopsis mutants with altered root caps. The primary root of lines of the fez-2 mutant, which has fewer root-cap cell layers and a more pointed root cap than wild-type roots, showed impaired penetration ability. Conversely, smb-3 roots, which display a rectangular-shaped cap, showed enhanced penetration abilities. These results, which contradict our original hypothesis, reveal a role for resistance to buckling in determining root penetration abilities.

Activities of Chromatin Remodeling Factors and Histone Chaperones and Their Effects in Root Apical Meristem Development.

Eukaryotic genes are packaged into dynamic but stable chromatin structures to deal with transcriptional reprogramming and inheritance during development. Chromatin remodeling factors and histone chaperones are epigenetic factors that target nucleosomes and/or histones to establish and maintain proper chromatin structures during critical physiological processes such as DNA replication and transcriptional modulation. Root apical meristems are vital for plant root development. Regarding the well-characterized transcription factors involved in stem cell proliferation and differentiation, there is increasing evidence of the functional implications of epigenetic regulation in root apical meristem development. In this review, we focus on the activities of chromatin remodeling factors and histone chaperones in the root apical meristems of the model plant species Arabidopsis and rice.

Identification of Periplasmic Root-Cap Mucilage in Developing Columella Cells of Arabidopsis thaliana.

Plant roots secrete various substances with diverse functions against both plants and microbes in the rhizosphere. A major secretory substance is root-cap mucilage, whose functions have been well characterized, albeit mainly in crops. However, little is currently known about the developmental mechanisms of root-cap mucilage. Here, we show the accumulation and extrusion of root-cap mucilage in Arabidopsis. We found propidium iodide (PI) stainable structures between the plasma membrane and cell wall in the sixth layer of columella cells (c6) from the quiescent center. Ruthenium red staining and PI staining with calcium ions suggested that the structure comprises in part pectin polysaccharides. Electron microscopy revealed that the structure had a meshwork of electron-dense filaments that resembled periplasmic mucilage in other plants. In the c6 cells, we also observed many large vesicles with denser meshwork filaments to periplasmic mucilage, which likely mediate the transport of mucilage components. Extruded mucilage was observed outside a partially degraded cell wall in the c7 cells. Moreover, we found that the Class IIB NAC transcription factors BEARSKIN1 (BRN1) and BRN2, which are known to regulate the terminal differentiation of columella cells, were required for the efficient accumulation of root-cap mucilage in Arabidopsis. Taken together, our findings reveal the accumulation of and dynamic changes in periplasmic mucilage during columella cell development in Arabidopsis.

Control of root cap maturation and cell detachment by BEARSKIN transcription factors in Arabidopsis.

The root cap supports root growth by protecting the root meristem, sensing gravity and interacting with the rhizosphere through metabolite secretion and cell dispersal. Sustained root cap functions therefore rely on balanced proliferation of proximal stem cells and regulated detachment of distal mature cells. Although the gene regulatory network that governs stem cell activity in the root cap has been extensively studied in Arabidopsis, the mechanisms by which root cap cells mature and detach from the root tip are poorly understood. We performed a detailed expression analysis of three regulators of root cap differentiation, SOMBRERO, BEARSKIN1 and BEARSKIN2, and identified their downstream genes. Our results indicate that expression of BEARSKIN1 and BEARSKIN2 is associated with cell positioning on the root surface. We identified a glycosyl hydrolase 28 (GH28) family polygalacturonase (PG) gene as a direct target of BEARSKIN1. Overexpression and loss-of-function analyses demonstrated that the protein encoded by this PG gene facilitates cell detachment. We thus revealed a molecular link between the key regulators of root cap differentiation and the cellular events underlying root cap-specific functions.

Tracking developmentally regulated post-synthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes.

Polysaccharides are major components of extracellular matrices and are often extensively modified post-synthetically to suit local requirements and developmental programmes. However, our current understanding of the spatiotemporal dynamics and functional significance of these modifications is limited by a lack of suitable molecular tools. Here, we report the development of a novel non-immunological approach for producing highly selective reciprocal oligosaccharide-based probes for chitosan (the product of chitin deacetylation) and for demethylesterified homogalacturonan. Specific reciprocal binding is mediated by the unique stereochemical arrangement of oppositely charged amino and carboxy groups. Conjugation of oligosaccharides to fluorophores or gold nanoparticles enables direct and rapid imaging of homogalacturonan and chitosan with unprecedented precision in diverse plant, fungal and animal systems. We demonstrated their potential for providing new biological insights by using them to study homogalacturonan processing during Arabidopsis thaliana root cap development and by analyzing sites of chitosan deposition in fungal cell walls and arthropod exoskeletons.

Haematopoietic effects of Angelica sinensis root cap polysaccharides against lisinopril-induced anaemia in albino rats.

CONTEXT: Angelica sinensis L. (Umbelliferae) has medicinal properties. OBJECTIVES: The present study evaluates the haematopoietic effects of A. sinensis polysaccharides (ASP) against lisinopril-induced anaemia. MATERIALS AND METHODS: Thirty healthy adult male albino rats were randomly divided into five groups (n = 6). Group I was control group. Group II was treated with angiotensin-converting enzyme inhibitor (ACEI, 20 mg/kg/day) to induce anaemia. In group III, erythropoietin (EPO, 100 IU/kg/each) was administered in combination with ACEI. Group IV was treated with ASP (1 g/kg/day), extracted from A. sinensis root caps. In Group V, ASP (1 g/kg/day) was treated with ACEI. After 28 days, blood and tissue samples were collected for haematological and histopathological analysis, respectively. RESULTS: The results showed that ACEI significantly reduced the haemoglobin (Hb, 10.0 g/dL), packed cell volume (PCV, 39.5%), red blood cells (RBCs, 6.2 million/mm3), mean corpuscular volume (MCV, 53.5 fL) and mean corpuscular haemoglobin (MCH, 16.2 pg/cell) values. In the group treated with ASP, the Hb (13.7 g/dL) and RBCs (7.8 million/mm3) increased significantly (p < 0.05). The combination of ASP and ACEI led to the significant (p < 0.05) reduction in Hb (10.7 g/dL), PCV (33.3%), RBCs (6.0 million/mm3), MCV (54.42 fL) and MCH (16.44 pg/cell) values. While histopathological examination of the liver and kidney cells showed a mild degree of toxicity in the ASP-treated group. CONCLUSION: ASP has a potentiating effect on haematological parameters when given alone. However, when administered simultaneously with lisinopril, it showed an unfavourable effect with more complicated anaemia so it should not be used with ACEIs.

The build-up of osmotic stress responses within the growing root apex using kinematics and RNA-sequencing.

Molecular regulation of growth must include spatial and temporal coupling of cell production and cell expansion. The underlying mechanisms, especially under environmental challenge, remain obscure. Spatial patterns of cell processes make the root apex well suited to deciphering stress signaling pathways, and to investigating both processes. Kinematics and RNA-sequencing were used to analyze the immediate growth response of hydroponically grown Populus nigra cuttings submitted to osmotic stress. About 7400 genes and unannotated transcriptionally active regions were differentially expressed between the division and elongation zones. Following the onset of stress, growth decreased sharply, probably due to mechanical effects, before recovering partially. Stress impaired cell expansion over the apex, progressively shortened the elongation zone, and reduced the cell production rate. Changes in gene expression revealed that growth reduction was mediated by a shift in hormone homeostasis. Osmotic stress rapidly elicited auxin, ethylene, and abscisic acid. When growth restabilized, transcriptome remodeling became complex and zone specific, with the deployment of hormone signaling cascades, transcriptional regulators, and stress-responsive genes. Most transcriptional regulations fit growth reduction, but stress also promoted expression of some growth effectors, including aquaporins and expansins Together, osmotic stress interfered with growth by activating regulatory proteins rather than by repressing the machinery of expansive growth.

Root cap-dependent gravitropic U-turn of maize root requires light-induced auxin biosynthesis via the YUC pathway in the root apex.

Gravitropism refers to the growth or movement of plants that is influenced by gravity. Roots exhibit positive gravitropism, and the root cap is thought to be the gravity-sensing site. In some plants, the root cap requires light irradiation for positive gravitropic responses. However, the mechanisms regulating this phenomenon are unknown. We herein report that maize roots exposed to white light continuously for ≥1-2h show increased indole-3-acetic acid (IAA) levels in the root tips, especially in the transition zone (1-3mm from the tip). Treatment with IAA biosynthesis inhibitors yucasin and l-kynurenine prevented any increases in IAA content and root curvature under light conditions. Analyses of the incorporation of a stable isotope label from tryptophan into IAA revealed that some of the IAA in roots was synthesized in the root apex. Furthermore, Zmvt2 and Zmyuc gene transcripts were detected in the root apex. One of the Zmyuc genes (ZM2G141383) was up-regulated by light irradiation in the 0-1mm tip region. Our findings suggest that IAA accumulation in the transition zone is due to light-induced activation of Zmyuc gene expression in the 0-1mm root apex region. Light-induced changes in IAA levels and distributions mediate the maize root gravitropic U-turn.

Suppression of Arabidopsis protophloem differentiation and root meristem growth by CLE45 requires the receptor-like kinase BAM3.

Peptide signaling presumably occupies a central role in plant development, yet only few concrete examples of receptor-ligand pairs that act in the context of specific differentiation processes have been described. Here we report that second-site null mutations in the Arabidopsis leucine-rich repeat receptor-like kinase gene barely any meristem 3 (BAM3) perfectly suppress the postembryonic root meristem growth defect and the associated perturbed protophloem development of the brevis radix (brx) mutant. The roots of bam3 mutants specifically resist growth inhibition by the CLAVATA3/ENDOSPERM SURROUNDING REGION 45 (CLE45) peptide ligand. WT plants transformed with a construct for ectopic overexpression of CLE45 could not be recovered, with the exception of a single severely dwarfed and sterile plant that eventually died. By contrast, we obtained numerous transgenic bam3 mutants transformed with the same construct. These transgenic plants displayed a WT phenotype, however, supporting the notion that CLE45 is the likely BAM3 ligand. The results correlate with the observation that external CLE45 application represses protophloem differentiation in WT, but not in bam3 mutants. BAM3, BRX, and CLE45 are expressed in a similar spatiotemporal trend along the developing protophloem, up to the end of the transition zone. Induction of BAM3 expression upon CLE45 application, ectopic overexpression of BAM3 in brx root meristems, and laser ablation experiments suggest that intertwined regulatory activity of BRX, BAM3, and CLE45 could be involved in the proper transition of protophloem cells from proliferation to differentiation, thereby impinging on postembryonic growth capacity of the root meristem.

Root tip contact with low-phosphate media reprograms plant root architecture.

Plant roots are able to sense soil nutrient availability. In order to acquire heterogeneously distributed water and minerals, they optimize their root architecture. One poorly understood plant response to soil phosphate (P(i)) deficiency is a reduction in primary root growth with an increase in the number and length of lateral roots. Here we show that physical contact of the Arabidopsis thaliana primary root tip with low-P(i) medium is necessary and sufficient to arrest root growth. We further show that loss-of-function mutations in Low Phosphate Root1 (LPR1) and its close paralog LPR2 strongly reduce this inhibition. LPR1 was previously mapped as a major quantitative trait locus (QTL); the molecular origin of this QTL is explained by the differential allelic expression of LPR1 in the root cap. These results provide strong evidence for the involvement of the root cap in sensing nutrient deficiency, responding to it, or both. LPR1 and LPR2 encode multicopper oxidases (MCOs), highlighting the essential role of MCOs for plant development.

Novel roles of hydrogen peroxide (H2O2) in regulating pectin synthesis and demethylesterification in the cell wall of rice (Oryza sativa) root tips.

Hydrogen peroxide (H2O2) has been reported to increase lignin formation, enhance cell wall rigidification, restrict cell expansion and inhibit root elongation. However, our results showed that it not only inhibited rice (Oryza sativa) root elongation, but also increased root diameter. No study has reported how and why H2O2 increases cell expansion and root diameter. Exogenous H2O2 and its scavenger 4-hydroxy-Tempo were applied to confirm the roles of H2O2. Immunofluorescence, fluorescence probe, ruthenium red staining, histological section and spectrophotometry were used to monitor changes in the degree of pectin methylesterification, pectin content, pectin methylesterase (PME) activity and H2O2 content. Exogenous H2O2 inhibited root elongation, but increased cell expansion and root diameter significantly. H2O2 not only increased the region of pectin synthesis and pectin content in root tips, but also increased PME activity and pectin demethylesterification. The scavenger 4-hydroxy-Tempo reduced root H2O2 content and recovered H2O2-induced increases in cell expansion and root diameter by inhibiting pectin synthesis, PME activity and pectin demethylesterification. H2O2 plays a novel role in the regulation of pectin synthesis, PME activity and pectin demethylesterification. H2O2 increases cell expansion and root diameter by increasing pectin content and demethylesterification.

The root cap: a short story of life and death.

Over 130 years ago, Charles Darwin recognized that sensory functions in the root tip influence directional root growth. Modern plant biology has unravelled that many of the functions that Darwin attributed to the root tip are actually accomplished by a particular organ-the root cap. The root cap surrounds and protects the meristematic stem cells at the growing root tip. Due to this vanguard position, the root cap is predisposed to receive and transmit environmental information to the root proper. In contrast to other plant organs, the root cap shows a rapid turnover of short-lived cells regulated by an intricate balance of cell generation, differentiation, and degeneration. Thanks to these particular features, the root cap is an excellent developmental model system, in which generation, differentiation, and degeneration of cells can be investigated in a conveniently compact spatial and temporal frame. In this review, we give an overview of the current knowledge and concepts of root cap biology, focusing on the model plant Arabidopsis thaliana.

The dual effects of root-cap exudates on nematodes: from quiescence in plant-parasitic nematodes to frenzy in entomopathogenic nematodes.

To defend themselves against herbivores and pathogens, plants produce numerous secondary metabolites, either constitutively or de novo in response to attacks. An intriguing constitutive example is the exudate produced by certain root-cap cells that can induce a state of reversible quiescence in plant-parasitic nematodes, thereby providing protection against these antagonists. The effect of such root exudates on beneficial entomopathogenic nematodes (EPNs) remains unclear, but could potentially impair their use in pest management programmes. We therefore tested how the exudates secreted by green pea (Pisum sativum) root caps affect four commercial EPN species. The exudates induced reversible quiescence in all EPN species tested. Quiescence levels varied with the green pea cultivars tested. Notably, after storage in root exudate, EPN performance traits were maintained over time, whereas performances of EPNs stored in water rapidly declined. In sharp contrast to high concentrations, lower concentrations of the exudate resulted in a significant increase in EPN activity and infectiousness, but still reduced the activity of two plant-parasitic nematode species. Our study suggests a finely tuned dual bioactivity of the exudate from green pea root caps. Appropriately formulated, it can favour long-term storage of EPNs and boost their infectiousness, while it may also be used to protect plants from plant-parasitic nematodes.

PCaP2 regulates nuclear positioning in growing Arabidopsis thaliana root hairs by modulating filamentous actin organization.

KEY MESSAGE: PCaP2 plays a key role in maintaining the nucleus at a relatively fixed distance from the apex during root hair growth by modulating actin filaments. During root hair growth, the nucleus localizes at a relatively fixed distance from the apex. In Arabidopsis thaliana, the position of the nucleus is mainly dependent on the configuration of microfilaments (filamentous actin). However, the mechanisms underlying the regulation of actin dynamics and organization for nuclear positioning are largely unknown. In the present study, we demonstrated that plasma membrane-associated Ca(2+) binding protein 2 (PCaP2) influences the position of the nucleus during root hair growth. Abnormal expression of PCaP2 in pcap2 and PCaP2 over-expression plants led to the disorganization of actin filaments, rather than microtubules, in the apex and sub-apical regions of root hairs, which resulted in aberrant root hair growth patterns and misplaced nuclei. Analyses using a PCaP2 mutant protein revealed that actin-severing activity is essential for the function of PCaP2 in root hairs. We demonstrated that PCaP2 plays a key role in maintaining nuclear position in growing root hairs by modulating actin filaments.

The receptor-like kinases GSO1 and GSO2 together regulate root growth in Arabidopsis through control of cell division and cell fate specification.

BACKGROUND: The root apical meristem of Arabidopsis is established post-embryonically as the main source of root cells, and its activity is maintained by complex bidirectional signaling between stem cells and mature cells. The receptor-like kinases GASSHO1 (GSO1) and GSO2 have been shown to regulate aerial epidermal function and seedling growth in Arabidopsis. RESULTS: Here we show that gso1; gso2 seedlings also have root growth and patterning defects. Analyses of mutant root morphology indicate abnormal numbers of cells in longitudinal files and radial cell layers, as well as aberrant stem cell division planes. gso1; gso2 double mutants misexpress markers for stem cells and differentiated root cell types. In addition, gso1; gso2 root growth defects, but not marker missexpression or patterning phenotypes, are rescued by growth on media containing metabolizable sugars. CONCLUSIONS: We conclude that GSO1 and GSO2 function together in intercellular signaling to positively regulate cell proliferation, differentiation of root cell types, and stem cell identity. In addition, GSO1 and GSO2 control seedling root growth by modulating sucrose response after germination.

Genome-wide transcriptome dissection of the rice root system: implications for developmental and physiological functions.

The root system is a crucial determinant of plant growth potential because of its important functions, e.g. uptake of water and nutrients, structural support and interaction with symbiotic organisms. Elucidating the molecular mechanism of root development and functions is therefore necessary for improving plant productivity, particularly for crop plants, including rice (Oryza sativa). As an initial step towards developing a comprehensive understanding of the root system, we performed a large-scale transcriptome analysis of the rice root via a combined laser microdissection and microarray approach. The crown root was divided into eight developmental stages along the longitudinal axis and three radial tissue types at two different developmental stages, namely: epidermis, exodermis and sclerenchyma; cortex; and endodermis, pericycle and stele. We analyzed a total of 38 microarray data and identified 22,297 genes corresponding to 17,010 loci that showed sufficient signal intensity as well as developmental- and tissue type-specific transcriptome signatures. Moreover, we clarified gene networks associated with root cap function and lateral root formation, and further revealed antagonistic and synergistic interactions of phytohormones such as auxin, cytokinin, brassinosteroids and ethylene, based on the expression pattern of genes related to phytohormone biosynthesis and signaling. Expression profiling of transporter genes defined not only major sites for uptake and transport of water and nutrients, but also distinct signatures of the radial transport system from the rhizosphere to the xylem vessel for each nutrient. All data can be accessed from our gene expression profile database, RiceXPro (http://ricexpro.dna.affrc.go.jp), thereby providing useful information for understanding the molecular mechanisms involved in root system development of crop plants.