Formation and separation of root border cells

Plant roots release a large number of border cells into the rhizosphere, which are believed to play a key role in root development and health. The formation and loss of these cells from the root cap region is a developmentally regulated process that is also controlled by phytohormones and environmental factors. The separation of border cells involves the complete dissociation of individual cells from each other and from root tissue. This process requires the activity of cell wall-degrading enzymes that solubilize the cell wall connections between cells. We present and discuss the solubilization process with an emphasis on pectin-degrading enzymes as well as the recently discovered root border-like cells of Arabidopsis thaliana.     

Extracellular proteins in pea root tip and border cell exudates

Newly generated plant tissue is inherently sensitive to infection. Yet, when pea (Pisum sativum) roots are inoculated with the pea pathogen, Nectria haematococca, most newly generated root tips remain uninfected even though most roots develop lesions just behind the tip in the region of elongation. The resistance mechanism is unknown but is correlated spatially with the presence of border cells on the cap periphery. Previously, an array of >100 extracellular proteins was found to be released while border cell separation proceeds. Here we report that protein secretion from pea root caps is induced in correlation with border cell separation. When this root cap secretome was proteolytically degraded during inoculation of pea roots with N. haematococca, the percentage of infected root tips increased from 4% +/- 3% to 100%. In control experiments, protease treatment of conidia or roots had no effect on growth and development of the fungus or the plant. A complex of >100 extracellular proteins was confirmed, by multidimensional protein identification technology, to comprise the root cap secretome. In addition to defense-related and signaling enzymes known to be present in the plant apoplast were ribosomal proteins, 14-3-3 proteins, and others typically associated with intracellular localization but recently shown to be extracellular components of microbial biofilms. We conclude that the root cap, long known to release a high molecular weight polysaccharide mucilage and thousands of living cells into the incipient rhizosphere, also secretes a complex mixture of proteins that appear to function in protection of the root tip from infection.     

Root border-like cells of Arabidopsis. Microscopical characterization and role in the interaction with rhizobacteria

Plant roots of many species produce thousands of cells that are released daily into the rhizosphere. These cells are commonly termed border cells because of their major role in constituting a biotic boundary layer between the root surface and the soil. In this study, we investigated the occurrence and ultrastructure of such cells in Arabidopsis (Arabidopsis thaliana) using light and electron microscopy coupled to high-pressure freezing. The secretion of cell wall molecules including pectic polysaccharides and arabinogalactan-proteins (AGPs) was examined also using immunofluorescence microscopy and a set of anticarbohydrate antibodies. We show that root tips of Arabidopsis seedlings released cell layers in an organized pattern that differs from the rather randomly dispersed release observed in other plant species studied to date. Therefore, we termed such cells border-like cells (BLC). Electron microscopical results revealed that BLC are rich in mitochondria, Golgi stacks, and Golgi-derived vesicles, suggesting that these cells are actively engaged in secretion of materials to their cell walls. Immunocytochemical data demonstrated that pectins as well as AGPs are among secreted material as revealed by the high level of expression of AGP-epitopes. In particular, the JIM13-AGP epitope was found exclusively associated with BLC and peripheral cells in the root cap region. In addition, we investigated the function of BLC and root cap cell AGPs in the interaction with rhizobacteria using AGP-disrupting agents and a strain of Rhizobium sp. expressing a green fluorescent protein. Our findings demonstrate that alteration of AGPs significantly inhibits the attachment of the bacteria to the surface of BLC and root tip.     

Inducible expression of <Emphasis Type="Italic">Pisum sativum</Emphasis> xyloglucan fucosyltransferase in the pea root cap meristem,and effects of antisense mRNA expression on root cap cell wall structural integrity

Mitosis and cell wall synthesis in the legume root cap meristem can be induced and synchronized by the nondestructive removal of border cells from the cap periphery. Newly synthesized cells can be examined microscopically as they differentiate progressively during cap development, and ultimately detach as a new population of border cells. This system was used to demonstrate that Pisum sativum L. fucosyl transferase (PsFut1) mRNA expression is strongly expressed in root meristematic tissues, and is induced >2-fold during a 5-h period when mitosis in the root cap meristem is increased. Expression of PsFut1 antisense mRNA in pea hairy roots under the control of the CaMV35S promoter, which exhibits meristem localized expression in pea root caps, resulted in a 50-60% reduction in meristem localized endogenous PsFut1 mRNA expression measured using whole mount in situ hybridization. Changes in gross levels of cell wall fucosylated xyloglucan were not detected, but altered surface localization patterns were detected using whole mount immunolocalization with CCRC-M1, an antibody that recognizes fucosylated xyloglucan. Emerging hairy roots expressing antisense PsFut1 mRNA appeared normal macroscopically but scanning electron microscopy of tissues with altered CCRC-M1 localization patterns revealed wrinkled, collapsed cell surfaces. As individual border cells separated from the cap periphery, cell death occurred in correlation with extrusion of cellular contents through breaks in the wall.     

Correlation of Pectolytic Enzyme Activity with the Programmed Release of Cells from Root Caps of Pea (Pisum sativum)

In many plant species, the daily release of hundreds to thousands of healthy cells from the root cap into the soil is a normal process, whose function is unknown. We studied the separation of the cells in pea (Pisum sativum) using an aeroponic system in which separated cells were retained on the root until they were washed off for counting. We found that cell separation is a developmentally regulated, temperature-sensitive process that appears to be regulated independently of root growth. No cells were released from very young roots. When plants were grown aeroponically, cell numbers increased with increasing root length to a mean of 3400 cells per root, at which point the release of new cells ceased. The process could be reset and synchronized by washing the root in water to remove shed cells. Cell separation from the root cap was correlated with pectolytic enzyme activity in root cap tissue. Because these cells that separate from the root cap ensheath the root as it grows and thus provide a cellular interface between the root surface and the soil, we propose to call the cells “root border cells.”     

Cloning of genes whose expression is correlated with mitosis and localized in dividing cells in root caps of Pisum sativum L.

Removal of border cells from pea roots synchronizes and induces root cap cell division, wall biogenesis and differentiation. Three messages which are expressed differentially in such induced root caps have been cloned. Sequence analyses showed that the PsHRGP1-encoded protein has high homology with a hydroxyproline-rich glycoprotein. The PsCaP23-encoded protein has high homology with an alfalfa callus protein or translationally controlled human or mouse tumor protein P23. The PsRbL41-encoded protein has high homology with a highly basic 60S ribosomal protein L41. In situ hybridization showed that PsHRGP1, PsCaP23 and PsRbL41 messages are localized within dividing cells of the root cap. PsHRGP1 is highly expressed in uninduced root caps, but its message is repressed by 10–11 times as soon as cell division and differentiation begin. Expression of PsHRGP1 recovers to higher than (180%) its initial level in 30 min. PsHRGP1 is root-specific. PsCaP23 and PsRbL41 messages increase ca. 3-fold within 15 min after root cap induction. All three genes represent small families of 3–5 closely related genes in the pea genome.     

边缘细胞对荞麦根尖铝毒的防护效应和对细胞壁多糖的影响

以2个荞麦(Fygopyrum esculentum Moench)基因型‘江西荞麦’(耐性)和‘内蒙荞麦’(敏感)为材料,采用悬空培养(保持边缘细胞附着于根尖和去除根尖边缘细胞),研究边缘细胞对根尖铝毒的防护效应以及对细胞壁多糖组分的影响。结果表明,铝毒抑制荞麦根系伸长,导致根尖Al积累。去除边缘细胞的根伸长抑制率和根尖Al含量高于保留边缘细胞的根。去除边缘细胞使江西荞麦和内蒙荞麦根尖的酸性磷酸酶(APA)活性显著升高,前者在铝毒下增幅更大。同时,铝毒胁迫下去除边缘细胞的根尖果胶甲酯酶(PME)活性和细胞壁果胶、半纤维素1、半纤维素2含量显著高于保留边缘细胞的酶活性和细胞壁多糖含量。表明边缘细胞对荞麦根尖的防护效应,与其阻止Al的吸收,降低根尖细胞壁多糖含量及提高酸性磷酸酶活性有关,以此缓解Al对根伸长的抑制。     

Meristem-Specific Suppression of Mitosis and a Global Switch in Gene Expression in the Root Cap of Pea by Endogenous Signals

Two functionally distinct sets of meristematic cells exist within root tips of pea (Pisum sativum): the root apical meristem, which gives rise to the body of the root; and the root cap meristem, which gives rise to cells that differentiate progressively through the cap and separate ultimately from its periphery as border cells. When a specific number of border cells has accumulated on the root cap periphery, mitosis within the root cap meristem, but not the apical meristem, is suppressed. When border cells are removed by immersion of the root tip in water, a transient induction of mitosis in the root cap meristem can be detected starting within 5 min. A corresponding switch in gene expression throughout the root cap occurs in parallel with the increase in mitosis, and new border cells begin to separate from the root cap periphery within 1 h. The induction of renewed border cell production is inhibited by incubating root tips in extracellular material released from border cells. The results are consistent with the hypothesis that operation of the root cap meristem and consequent turnover of the root cap is self-regulated by a signal from border cells.     

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.     

Effect of pectin methylesterase gene expression on pea root development.

Expression of an inducible gene with sequences common to genes encoding pectin methylesterase (PME) was found to be tightly correlated, both spatially and temporally, with border cell separation in pea root caps. Partial inhibition of the gene's expression by antisense mRNA in transgenic pea hairy roots prevented the normal separation of root border cells from the root tip into the external environment. This phenotype was correlated with an increase in extracellular pH, reduced root elongation, and altered cellular morphology. The translation product of the gene exhibited PME activity in vitro. These results are consistent with the long-standing hypothesis that the demethylation of pectin by PME plays a key role in cell wall metabolism.     

Synthesis and oxidative insolubilization of cell-wall proteins during osmotic stress

The cell walls in the new white roots of jack pine (Pinus banksiana Lamb.) were observed to constrict around the shrinking protoplast of osmotically stressed roots, and pressure was maintained via an apparent adjustment of cell-wall size and elasticity. These elastic alterations of the cell wall permitted the root cells to maintain full turgor despite the loss of most of the water in the tissue. The constriction of the root cell wall around the dehydrating protoplasts to maintain turgor may reflect changes in cell wall structure. We found that these shrinking root cells synthesize and secrete into the intercellular fluid a set of proteins. These proteins become tightly associated (i.e. guanidine HCl- and sodium dodecyl sulfate-insoluble) with the cell wall but can be released from the matrix, after briefly boiling in 0.1% sodium dodecyl sulfate, by the combination of guanidine HCl, CaCl₂ and dithiothreitol. However, these cell-wall proteins became insoluble with time. The proteins could subsequently be destructively extracted from the wall with acid NaClO₂ treatments. After these proteins were incorporated into the cell walls, the roots adopted a new, smaller maximal tissue volume and elastic coefficients returned to normal levels. Received: 8 July 1998 / Accepted: 19 November 1998     

Stimulation of border cell production in response to increased carbon dioxide levels

Field soil atmospheres have higher CO(2) and lower O(2) concentrations compared with ambient atmosphere, but little is known about the impact of such conditions on root exudation patterns. We used altered levels of CO(2) and O(2) relative to ambient conditions to examine the influence of the atmosphere on the production of root border cells by pea (Pisum sativum) root tips. During germination, atmospheres with high CO(2) and low O(2) inhibited root development and border cell separation in pea seedlings. Later in development, the same atmospheric composition stimulated border cell separation without significantly influencing root growth. Increased CO(2), not low O(2), was responsible for the observed stimulation of border cell number. High CO(2) apparently can override endogenous signals that regulate the number of border cells released from pea roots into the rhizosphere. The same conditions that stimulated border cell production in pea had no such effect in alfalfa (Medicago sativa).     

Identification and characterization of a rhizosphere β-galactosidase from Pisum sativum L.

Plant enzyme activities in the rhizosphere potentially are a resource for improved plant nutrition, soil fertility, bioremediation, and disease resistance. Here we report that a border cell specific β-galactosidase is secreted into the acidic extracellular environment surrounding root tips of pea, as well as bean, alfalfa, barrel medic, sorghum, and maize. No enzyme activity was detected in radish and Arabidopsis, species that do not produce viable border cells. The secreted enzyme activity was inhibited by galactose and 2-phenylethyl 1-thio-β-d-galactopyranoside (PETG) at concentrations that altered root growth without causing cell death. A tomato galactanase encoding gene was used as a probe to isolate a full length pea cDNA clone (BRDgal1) from a root cap-border cell cDNA library. Southern blot analysis using full length BRDgal1 as a probe revealed 1–2 related sequences within the pea genome. BRDgal1 mRNA expression was analysed by whole mount in situ hybridization (WISH) and found to occur in the outermost peripheral layer of the cap and in suspensions of detached border cells. No expression was detected within the body of the root cap. Repeated efforts to develop viable hairy root clones expressing BRDgal1 antisense mRNA under the control of the CaMV35S promoter, whose expression in the root cap is limited to cells at the root cap periphery only during root emergence, were unsuccessful. These data suggest that altered expression of this enzyme is deleterious to early root development. The first two authors contributed equally to the completion of this project.     

ADVENTITIOUS ROOT DEVELOPMENT FROM THE COLEOPTILAR NODE IN ZEA MAYS L.

Department of Botany and Bacteriology, University of Arkansas, Fayetteville, Arkansas 72701 Zea mays L. root development from the coleoptilar node was observed by light and electron microscopy. Roots developed opposite collateral vascular bundles in the coleoptilar nodal region. Three distinct histogens (stelar, cortical-protoderm, and root cap) became evident in early development. In median sections of the young roots, root cap and cortical regions formed a “hat” configuration over the stelar region. As the root matured, this “hat” developed centripetally to encapsulate the stelar region. Central core cells of the root cap were characterized by having numerous dictyosomes, amyloplasts, vacuoles, and thin cell walls. As these cells matured into outer or peripheral cap cells, the Golgi vesicles became hypertrophied. These hypertrophied vesicles contained a granular PAS-positive material which accumulated between the plasma membrane and the cell wall and formed a thick layer. As the PAS-positive material passed through the cell wall, it changed to a fibrillar texture. A PAS-positive material similar to that in the outer root cap cells was found adjacent to the outer walls of the protodermal cells. In median sections, PAS-positive material was not present in the promeristem region. Root cap cells as well as parent cortical cells were crushed as the young root forced its way through the parent tissue.     

Changes in cell wall ultrastructure induced by sudden flooding at 25{degrees}C in Pisum sativum (Fabaceae) primary roots

Cellular degeneration is essential for many developmental and stress acclimation processes. Undifferentiated parenchymatous cells in the central vascular cylinder of pea primary roots degenerate under hypoxic conditions created by flooding at temperatures >15°C, forming a long vascular cavity that seems to provide a conduit for longitudinal oxygen transport in the roots. We show that specific changes in the cell wall ultrastructure accompanied previously detected cytoplasmic and organellar degradation in the cavity-forming roots. The degenerating cells had thinner primary cell walls, less electron-dense middle lamellae, and less abundant cell wall homogalacturonans in altered patterns, compared to healthy cells of roots grown under cold, nonflooded conditions. Cellular breakdown and changes in wall ultrastructure, however, remained confined to cells within a 50-μm radius around the root center, even after full development of the cavity. Cells farther away maintained cellular integrity and had signs of wall synthesis, perhaps from tight regulation of wall metabolism over short distances. These observations suggest that the cell degeneration might involve programmed cell death. We also show that warm, nonflooded or cold, flooded conditions that typically do not induce vascular cavity formation can also induce variations in cell wall ultrastructure.     

Localization of repetitive proline-rich proteins in the extracellular matrix of pea root nodules

Summary Early responses of legume roots toRhizobium inoculation include new cell wall synthesis and induction of some putative wall protein genes. Although the predicted amino acid sequences of several early nodulins indicate that they encode proline-rich proteins (PRPs), the proteins have been neither isolated nor has their presence been demonstrated in cell walls. We have used polyclonal antibodies against PRP2 from soybean to identify and localize proline-rich proteins in pea nodules. On immunoblots, several PRPs were detected, ranging from less than 20 kDa to 110 kDa. Immunocytochemistry revealed that tissues of the vascular cylinder contained abundant PRPs, particularly in the secondary cell walls of xylem elements and phloem fibers. PRPs were also found within the primary wall of the nodule endodermis and within Casparian strips of the vascular endodermis. Of symbiotic importance, PRPs were a prominent component of the infection thread matrix in newly infected root cells and in nodules. PRPs were also secreted by cells in the uninfected nodule parenchyma, where they were found occluding intercellular spaces outside the middle lamella. Despite structural conservation among members of this class of cell wall proteins, PRPs were targeted to distinct layers of the extracellular matrix dependent upon cell type, and may thus play separate roles in the biology of plant cells. The putative functions and the potential for interactions between PRPs and other wall polymers are discussed.Abbreviations DTT dithiothreitol - EDTA ethylenediamine tetraacetate - GRP glycine-rich protein - PCR polymerase chain reaction - PGA polygalacturonic acid - PMSF phenylmethylsulfonyl fluoride - PRP proline-rich protein - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - Tris tris(hydroxylmethyl) aminomethane - Tween 20 polyoxyethylene sorbitan monolaurate Dedicated to the memory of Professor John G. Torrey     

The Arabidopsis SOS5 locus encodes a putative cell surface adhesion protein and is required for normal cell expansion

Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein-like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf.     

The production and release of living root cap border cells is a function of root apical meristem type in dicotyledonous angiosperm plants

BACKGROUND AND AIMS: The root apical meristems (RAM) of flowering plant roots are organized into recognizable pattern types. At present, there are no known ecological or physiological benefits to having one RAM organization type over another. Although there are phylogenetic distribution patterns in plant groups, the possible evolutionary advantages of different RAM organization patterns are not understood. Root caps of many flowering plant roots are known to release living border cells into the rhizosphere, where the cells are believed to have the capacity to alter conditions in the soil and to interact with soil micro-organisms. Consequently, high rates of border cell production may have the potential to benefit plant growth and development greatly, and to provide a selective advantage in certain soil environments. This study reports the use of several approaches to elucidate the anatomical and developmental relationships between RAM organization and border cell production. METHODS: RAM types from many species were compared with numbers of border cells released in those species. In addition, other species were grown, fixed and sectioned to verify their organization type and capacity to produce border cells. Root tips were examined microscopically to characterize their pattern and some were stained to determine the viability of root cap cells. KEY RESULTS: The first report of a correlation between RAM organization type and the production and release of border cells is provided: species exhibiting open RAM organization produce significantly more border cells than species exhibiting closed apical organization. Roots with closed apical organization release peripheral root cap cells in sheets or large groups of dead cells, whereas root caps with open organization release individual living border cells. CONCLUSIONS: This study, the first to document a relationship between RAM organization, root cap behaviour and a possible ecological benefit to the plant, may yield a framework to examine the evolutionary causes for the diversification of RAM organization types across taxa.