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.

Root extracellular traps versus neutrophil extracellular traps in host defence, a case of functional convergence?

The root cap releases cells that produce massive amounts of mucilage containing polysaccharides, proteoglycans, extracellular DNA (exDNA) and a variety of antimicrobial compounds. The released cells - known as border cells or border-like cells - and mucilage secretions form networks that are defined as root extracellular traps (RETs). RETs are important players in root immunity. In animals, phagocytes are some of the most abundant white blood cells in circulation and are very important for immunity. These cells combat pathogens through multiple defence mechanisms, including the release of exDNA-containing extracellular traps (ETs). Traps of neutrophil origin are abbreviated herein as NETs. Similar to phagocytes, plant root cap-originating cells actively contribute to frontline defence against pathogens. RETs and NETs are thus components of the plant and animal immune systems, respectively, that exhibit similar compositional and functional properties. Herein, we describe and discuss the formation, molecular composition and functional similarities of these similar but different extracellular traps.

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.

Xyloglucan and cellulose form molecular cross-bridges connecting root border cells in pea (Pisum sativum).

Pea (Pisum sativum) root cap releases a large number of living border cells that secrete abundant mucilage into the extracellular medium. Mucilage contains a complex mixture of polysaccharides, proteins and secondary metabolites important for its structure and function in defense. Unlike xyloglucan and cellulose, pectin and arabinogalactan proteins have been investigated in pea root and shown to be major components of border cell walls and mucilage. In this study, we investigated the occurrence of xyloglucan and cellulose in pea border cells and mucilage using cytochemical staining, immunocytochemistry and laser scanning confocal microscopy. Our data show that i) unlike cellulose, xyloglucan is highly present in the released mucilage as a dense fibrillary network enclosing border cells and ii) that xyloglucan and cellulose form molecular cross-bridges that tether cells and maintain them attached together. These findings suggest that secreted xyloglucan is essential for mucilage strengthening and border cell attachment and functioning.

Cytogenetics and morphological delimitation between three species of Passiflora L. (subgenus Distephana Cervi).

Several studies on cytogenetic characterisation of passion flowers are helpful to elucidate doubts about taxa relationships, delimitation and classification into more coherent groups based on karyomorphological data. Molecular and conventional cytogenetic techniques were applied to three Passiflora species with red flowers, P. coccinea, P. vitifolia and P. tholozanii, for species karyotype relationships. Additionally, for descriptive morphology, were used flowers, leaves and seeds. Results describe for the first time the karyomorphological and chromosome number (2n = 18) for P. tholozanii. anova was performed (P < 0.05) and statistical significance for average chromosome size (CV: 16.53%) between species. Genomic in situ hybridisation (GISH) proved relationships between P. coccinea and P. tholozanii, which suggests a common origin, however, we could not identify hybridisation between genomic probes from P. vitifolia in P. tholozanii chromosomes. Among the species analysed, P. tholozanii has great similarity in karyotypic and morphology to P. coccinea but not to P. vitifolia. We suggest the inclusion of P. tholozanii in the same subgenus and section as P. coccinea based on the similarity in karyomorphological and morphological traits between the species. Additionally, GISH might indicate a common or hybrid origin of P. tholozanii.

Aluminium-induced cell death requires upregulation of NtVPE1 gene coding vacuolar processing enzyme in tobacco (Nicotiana tabacum L.).

Cell death mechanism triggered by aluminium (Al) ion was investigated at root apex of tobacco (cultivar Bright Yellow) and in cultured tobacco cell line BY-2 derived from Bright Yellow, focusing on VPE genes (NtVPE1a, NtVPE1b, NtVPE2, NtVPE3). Cell death was detected as a loss of integrity of the plasma membrane by vital staining with fluorescein diacetate (in root apex) and Evans blue (in BY-2), respectively. At root apex, the upregulation of gene expression of VPE1a and VPE1b was observed significantly after 9h of Al exposure in parallel with an enhancement of cell death, while the upregulation of VPE2 and VPE3 were observed later. Similarly, in BY-2 cells, the upregulation of VPE1a and VPE1b and the enhancement of cell death were synchronously observed after 3-h exposure to Al, while the upregulation of VPE2 and VPE3 occurred later. RNA interference (RNAi) lines of each of the VPEs were constructed in BY-2 cells. Comparative studies between wild-type and the RNAi lines indicated that both Al-enhanced VPE activity and Al-induced cell death were significantly suppressed in the RNAi lines of VPE1 (dual suppressor of VPE1a and VPE1b), but not in the RNAi lines of VPE2 and that of VPE3. Taken together, we conclude that the upregulation of VPE1 gene expression and following enhancement of VPE activity under Al stress cause cell death in actively growing or elongating cells of tobacco.

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.

Cyclic programmed cell death stimulates hormone signaling and root development in Arabidopsis.

The plant root cap, surrounding the very tip of the growing root, perceives and transmits environmental signals to the inner root tissues. In Arabidopsis thaliana, auxin released by the root cap contributes to the regular spacing of lateral organs along the primary root axis. Here, we show that the periodicity of lateral organ induction is driven by recurrent programmed cell death at the most distal edge of the root cap. We suggest that synchronous bursts of cell death in lateral root cap cells release pulses of auxin to surrounding root tissues, establishing the pattern for lateral root formation. The dynamics of root cap turnover may therefore coordinate primary root growth with root branching in order to optimize the uptake of water and nutrients from the soil.

Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination.

Root tips of many plant species release a number of border, or border-like, cells that are thought to play a major role in the protection of root meristem. However, little is currently known on the structure and function of the cell wall components of such root cells. Here, we investigate the sugar composition of the cell wall of the root cap in two species: pea (Pisum sativum), which makes border cells, and Brassica napus, which makes border-like cells. We find that the cell walls are highly enriched in arabinose and galactose, two major residues of arabinogalactan proteins. We confirm the presence of arabinogalactan protein epitopes on root cap cell walls using immunofluorescence microscopy. We then focused on these proteoglycans by analyzing their carbohydrate moieties, linkages, and electrophoretic characteristics. The data reveal (1) significant structural differences between B. napus and pea root cap arabinogalactan proteins and (2) a cross-link between these proteoglycans and pectic polysaccharides. Finally, we assessed the impact of root cap arabinogalactan proteins on the behavior of zoospores of Aphanomyces euteiches, an oomycetous pathogen of pea roots. We find that although the arabinogalactan proteins of both species induce encystment and prevent germination, the effects of both species are similar. However, the arabinogalactan protein fraction from pea attracts zoospores far more effectively than that from B. napus. This suggests that root arabinogalactan proteins are involved in the control of early infection of roots and highlights a novel role for these proteoglycans in root-microbe interactions.

A cell-type-specific defect in border cell formation in the Acacia mangium root cap developing an extraordinary sheath of sloughed-off cells.

BACKGROUND AND AIMS: Root caps release border cells, which play central roles in microbe interaction and root protection against soil stresses. However, the number and connectivity of border cells differ widely among plant species. Better understanding of key border-cell phenotype across species will help define the total function of border cells and associated genes. METHODS: The spatio-temporal detachment of border cells in the leguminous tree Acacia mangium was investigated by using light and fluorescent microscopy with fluorescein diacetate, and their number and structural connectivity compared with that in soybean (Glycine max). KEY RESULTS: Border-like cells with a sheet structure peeled bilaterally from the lateral root cap of A. mangium. Hydroponic root elongation partially facilitated acropetal peeling of border-like cells, which accumulate as a sheath that covers the 0- to 4-mm tip within 1 week. Although root elongation under friction caused basipetal peeling, lateral root caps were minimally trimmed as compared with hydroponic roots. In the meantime, A. mangium columella caps simultaneously released single border cells with a number similar to those in soybean. CONCLUSIONS: These results suggest that cell type-specific inhibitory factors induce a distinct defective phenotype in single border-cell formation in A. mangium lateral root caps.

[Ultrastructure of statocytes and cells of distal elongation zone of Arabidopsis thaliana under clinorotation].

Results of the electron-microscopic investigation of root apices of Arabidopsis thaliana 3-, 5- and 7-days-old seedlings grown in the stationary conditions and under clinorotation are presented. It was shown the similarity in the root apex cell ultrastructure in control and under clinorotation. At the same time there were some differences in the ultrastructure of statocytes and the distal elongation zone under clinorotation. For the first time the sensitivity of ER-bodies, which are derivatives of GER and contain beta-glucosidase, to the influence of simulated microgravity was demonstrated by increased quantity and area of ER-bodies at the cell section as well as by higher variability of their form under clinorotation. A degree of these changes correlated with the duration of clinorotation. On the basis of experimental data a protective role of ER-bodies in adaptation of plants to microgravity is supposed.

[Micromorphometric analysis of root cap cells in Allium cepa].

A two-dimensional micromorphometric analysis of a root cap in Allium cepa included the measurement of the areas of about six thousand cell profiles on both longitudinal and transversal sections. Basing on the results of this analysis, quantitative descriptions of two cell populations from the columella and periferic part of the root cap have been created. The scheme of arrangement of root cap cells, located at a distance of 250 microm or less from a root tip, has been developed.

D'orenone blocks polarized tip growth of root hairs by interfering with the PIN2-mediated auxin transport network in the root apex.

SUMMARY: The C(18) ketone (5E,7E)-6-methyl-8-(2,6,6-trimethylcyclohex-1-enyl)octa-5,7-dien-2-one (D'orenone) has been postulated to be an early cleavage product of beta-carotene en route to trisporic acids; these act as morphogenetic factors during the sexual reproduction of zygomycetes. Here we report that D'orenone blocks the highly polarized tip growth of root hairs, causing tip growth to stop completely within a few minutes. Importantly, external auxin reverses the effects of D'orenone on root hairs. Further analysis revealed that D'orenone lowers the auxin concentration in trichoblasts via PIN2-mediated auxin efflux to below the critical levels essential for root hair growth. D'orenone specifically increases PIN2 protein abundance without affecting PIN2 transcripts, and the PIN2 expression domain enlarges and shifts basipetally, resulting in more active auxin transport. The observation that D'orenone does not interfere with the root hair growth in roots of null mutant lines provides additional evidence that PIN2 is its specific target.

[The growth and differentiation of root cap columella cells and the proper root grown in the stationary conditions and under clinorotation].

The results of light- and electron-microscopic investigations of root apices of Beta vulgaris 3-day-old seedlings grown in the stationary conditions and under clinorotation are presented. It was shown that ultrastructure and topography of organelles in root cap statocytes (graviperceptive cells) and in the cells of distal elongation zone clearly reflected the different direction in their growth and differentiation in space and time in dependence on specialization and functions. Cell growth and genetically determined differentiation occur similarly to control, although certain differences in ultrastructure are evident on metabolism changes.

Proteomics analysis of rice lesion mimic mutant (spl1) reveals tightly localized probenazole-induced protein (PBZ1) in cells undergoing programmed cell death.

Numerous reports have predicted/hypothesized a role for probenazole-induced protein (PBZ1) as a molecular marker in rice self-defense mechanism. However, the precise function of PBZ1 remains unknown. In the present study, we examined PBZ1 as a putative cell death marker in rice. For this, we focused our attention on a rice lesion mimic mutant (LMM), spotted leaf 1 ( spl1), which has been used to study the programmed cell death (PCD) phenomenon during lesion development in leaf. Using two-dimensional gel electrophoresis (2-DGE), 18 colloidal Coomassie brilliant blue stained protein spots were found to be differentially expressed in the leaves of spl1 mutant. After analysis of these spots by MALDI-TOF-MS, we identified the PBZ1 protein to be highly inducible in spl1. On the basis of these results, we proceeded to verify whether PBZ1 is highly expressed in the tissues undergoing PCD in rice. To do so, we performed immunoblot analysis and immunolocalization and used transgenic lines carrying the PBZ1 promoter fused with GFP. Results demonstrated that the expression levels and localizations of PBZ1 dramatically coincided with tissues undergoing PCD, namely, during leaf senescence, root aerenchyma formation, coleoptiles senescence, root cap, and seed aleurone layer. Furthermore, localization of the PBZ1 protein was also tightly correlated with TUNEL signal in the seed aleurone layer. As DNA fragmentation is a hallmark of PCD, this result clearly indicates a role for PBZ1 in rice tissues undergoing PCD. In conclusion, our results provide strong support for the hypothesis that PBZ1 is a molecular marker in rice defense response, and can serve as a novel potential marker for cell death/PCD in rice.

Ethylene responses in Arabidopsis seedlings include the reduction of curvature values in the root cap.

Recently, curvature was described as a new trait useful in the analysis of root apex shape. Treating the root profile as a geometric curve revealed that root apex curvature values are lower in ethylene-insensitive mutants (Cervantes E, Tocino A. Geometric analysis of Arabidopsis root apex reveals a new aspect of the ethylene signal transduction pathway in development. J Plant Physiol 2005;162:1038-45). This fact suggests that curvature is regulated by ethylene. In this work, we have determined the curvature values in embryonic roots of wild-type Columbia as well as in ethylene signal-transduction mutants, and found smaller values in embryos of the mutants. We also report on the evolution of root curvature during early development after seed germination. The line Lt16b that expresses GFP in the cell wall has allowed us to investigate the evolution of curvature values in three successive cell layers of seedling roots by confocal microscopy. Treatment of seedlings with norbornadiene resulted in lower curvature values. Our results show details illustrating the effect of ethylene in root curvature.

AXR4 is required for localization of the auxin influx facilitator AUX1.

The AUX1 and PIN auxin influx and efflux facilitators are key regulators of root growth and development. For root gravitropism to occur, AUX1 and PIN2 must transport auxin via the lateral root cap to elongating epidermal cells. Genetic studies suggest that AXR4 functions in the same pathway as AUX1. Here we show that AXR4 is a previously unidentified accessory protein of the endoplasmic reticulum (ER) that regulates localization of AUX1 but not of PIN proteins. Loss of AXR4 resulted in abnormal accumulation of AUX1 in the ER of epidermal cells, indicating that the axr4 agravitropic phenotype is caused by defective AUX1 trafficking in the root epidermis.

Auxin and ethylene interactions control mitotic activity of the quiescent centre, root cap size, and pattern of cap cell differentiation in maize.

Root caps (RCs) are the terminal tissues of higher plant roots. In the present study the factors controlling RC size, shape and structure were examined. It was found that this control involves interactions between the RC and an adjacent population of slowly dividing cells, the quiescent centre, QC. Using the polar auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA), the effects of QC activation on RC gene expression and border cell release was characterized. Ethylene was found to regulate RC size and cell differentiation, since its addition, or the inhibition of its synthesis, affected RC development. The stimulation of cell division in the QC following NPA treatment was reversed by ethylene, and quiescence was re-established. Moreover, inhibition of both ethylene synthesis and auxin polar transport triggered a new pattern of cell division in the root epidermis and led to the appearance of supernumerary epidermal cell files with cap-like characteristics. The data suggest that the QC ensures an ordered internal distribution of auxin, and thereby regulates not only the planes of growth and division in both the root apex proper and the RC meristem, but also regulates cell fate in the RC. Ethylene appears to regulate the auxin redistribution system that resides in the RC. Experiments with Arabidopsis roots also reveal that ethylene plays an important role in regulating the auxin sink, and consequently cell fate in the RC.

The promotion of gravitropism in Arabidopsis roots upon actin disruption is coupled with the extended alkalinization of the columella cytoplasm and a persistent lateral auxin gradient.

The actin cytoskeleton has been implicated in regulating plant gravitropism. However, its precise role in this process remains uncertain. We have shown previously that disruption of the actin cytoskeleton with Latrunculin B (Lat B) strongly promoted gravitropism in maize roots. These effects were most evident on a clinostat as curvature that would exceed 90 degrees despite short periods of horizontal stimulation. To probe further the cellular mechanisms underlying these enhanced gravity responses, we extended our studies to roots of Arabidopsis. Similar to our observations in other plant species, Lat B enhanced the response of Arabidopsis roots to gravity. Lat B (100 nm) and a stimulation time of 5-10 min were sufficient to induce enhanced bending responses during clinorotation. Lat B (100 nm) disrupted the fine actin filament network in different regions of the root and altered the dynamics of amyloplasts in the columella but did not inhibit the gravity-induced alkalinization of the columella cytoplasm. However, the duration of the alkalinization response during continuous gravistimulation was extended in Lat B-treated roots. Indirect visualization of auxin redistribution using the DR5:beta-glucuronidase (DR5:GUS) auxin-responsive reporter showed that the enhanced curvature of Lat B-treated roots during clinorotation was accompanied by a persistent lateral auxin gradient. Blocking the gravity-induced alkalinization of the columella cytoplasm with caged protons reduced Lat B-induced curvature and the development of the lateral auxin gradient. Our data indicate that the actin cytoskeleton is unnecessary for the initial perception of gravity but likely acts to downregulate gravitropism by continuously resetting the gravitropic-signaling system.

The evaluation of the genotoxicity of two commonly used food colors: Quinoline Yellow (E 104) and Brilliant Black BN (E 151).

Additives, especially colors, are in widespread use in the food industry. With the exception of the quinolines, food colors are relatively weak mutagens and are certified as safe additives despite reports that some people have allergic reactions to them. The number of food additives is still on the increase, and research on their potential mutagenic/carcinogenic activity in vivo is very expensive. Using two different cellular model systems, human lymphocytes in vitro and Vicia faba root tip meristems of in vivo, we evaluated the potential cytological and genotoxic effects of two dyes: Quinoline Yellow (E 104) and Brilliant Black BN (E 151). Two relatively new, very sensitive and rapid tests - the micronucleus and Comet assays - were used in this study. The data provided in this paper showed the genotoxic effects of the two analyzed food colors, and confirmed the diagnostic value of the MN and Comet assays for screening potentially genotoxic substances.