Uptake of Proteins by Plant Roots

The patterns of uptake of fluorescein-labelled lysozyme (Fl-lysozyme) by barley, maize, onion, tomato and vetch are similar as revealed by fluorescence microscopy. Penetration of the root cap and through the epidermis into the cortex increases with time of exposure and decreases with higher salt concentrations. In fact, one molar ethylammonium chloride can remove most of the absorbed protein from treated roots and the space observed to be stained by Fl-lysozyme in this manner can be visualized as “free space”. Results with sterile and non-sterile barley roots were indistinguishable. At low ionic strength, Fl-lysozyme can penetrate cells and complex with nucleoli. Such cell protoplasts appear “coagulated”. Uptake results with fluorescein per se were unlike those with protein. The uptake of a much larger molecule, ferritin, is confined to the epidermis and root cell walls. Localized, absorbed protein and root growth inhibition by basic proteins have yet to be related.     

Uptake of Lucifer Yellow CH into intact barley roots: Evidence for fluid-phase endocytosis

Intact barley (Hordeum vulgare L.) roots have been shown to take up the highly fluorescent dye Lucifer Yellow CH (LYCH) into their cell vacuoles. In the apical 1 cm of root tip, differentiating and dividing cells showed a prolific uptake of LYCH into their provacuoles. The LYCH was retained during fixation, apparently becoming bound to electron-dense material in the vacuoles. The dye freely entered the apoplast of roots in which the Casparian band was not developed, being taken up into the vacuoles of cells in both the cortex and stele. However, when LYCH was applied to a 1-cm zone approx. 6 cm behind the root tip the Casparian band on the radial walls of the endodermis completely prevented the dye from entering the cells of the stele, only the cell walls and vacuoles of the cortical cells taking up the dye. The inability of LYCH to cross the plasmalemma of the endodermal cells and enter the stele via the symplast substantiates previous claims that the dye is unable to cross the plasmalemma of plant cells. The results are discussed in the light of recent demonstrations that LYCH is a particularly effective marker for fluid-phase endocytosis in animal and yeast cells. A calculation of the energetic requirements for LYCH uptake into barley roots supports the contention that LYCH is taken up into the vacuoles of plant cells by fluid-phase endocytosis.Abbreviation LYCH Lucifer Yellow CH     

Entry of a basic protein, lysozyme, through the region of elongation in roots of Iasione montana

Ultrastructural effects of a basic protein, lysozyme, on rootcells of Iasione montana have been related to cellular mechanismsof root growth inhibition. Lysozyme is found to disrupt cellwalls and to disintegrate cellular membranes of elongating cells,but not membranes of mature and meristematic cells. Lysozymeseems to penetrate roots only in the region of elongation anddoes not affect meristematic cells; meristematic cells of Iasionemontana possess very thick peripheral cell walls covered byan electronopaque layer. As the cells elongate, primary cellwalls become thinner, the microfibrillar network becomes looserand the dense layer breaks off. Elongating cells are more vulnerableto penetration by lysozyme; however, as the cells mature, thereis a barrier to lysozyme formed that consists of secondary cellwalls; these appear to be a tightly arranged network of macromolecules. ¹ Present address: Department of Biology, Massachusetts Instituteof Technology, Cambridge, Massachusetts 02139, U.S.A. (Received November 29, 1974; )     

ENTRY OF BASIC MACROMOLECULES INTO BARLEY ROOTS

The entry of labelled calf-thymus histone, lysozyme, and poly-L-lysine into barley root tips was studied at concentrations which strongly inhibit root elongation. The macromolecules were suitably labelled and at these concentrations it was found, by autoradiography and fluorescence microscopy, that histone and lysozyme readily entered the roots and appeared to bind mainly to cell walls of the epidermis and cortex and to penetrate the cytoplasm occasionally. Except in cap cells, nuclei were rarely penetrated. Poly-L-lysine readily permeated cell walls and invaded cytoplasm and nuclei throughout the root tip. Some cells were damaged by contact with basic macromolecules, as evidenced by a change in appearance of protoplasts under phase contrast and by the inability of these same protoplasts to exclude labelled β-lactoglobulin. Such damage was restricted to cells in contact with the outer solution. Interior to the epidermis, development of many cells was inhibited without visible signs of damage. Evidence supports the conclusion that in the presence of polybasic polymers the integrity of cell membranes is altered, thereby allowing leakage of some cell constituents essential for normal development.     

PRPs localized to the middle lamellae are required for cortical tissue integrity in Medicago truncatula roots

Key message

A family of repetitive proline-rich proteins interact with acidic pectins and play distinct roles in legume root cell walls affecting cortical and vascular structure.

Abstract

A proline-rich protein (PRP) family, composed of tandemly repeated Pro-Hyp-Val-X-Lys pentapeptide motifs, is found primarily in the Leguminosae. Four distinct size classes within this family are encoded by seven tightly linked genes: MtPRP1, MtPRP2 and MtPRP3, and four nearly identical MtPRP4 genes. Promoter fusions to β-glucuronidase showed strong expression in the stele of hairy roots for all 4 PRP genes tested, with additional expression in the cortex for PRP1, PRP2 and PRP4. All except MtPRP4 are strongly expressed in non-tumorous roots, and secreted and ionically bound to root cell walls. These PRPs are absent from root epidermal cell walls, and PRP accumulation is highly localized within the walls of root cortical and vascular tissues. Within xylem tissue, PRPs are deposited in secondary thickenings where it is spatially exclusive to lignin. In newly differentiating xylem, PRPs are deposited in the regularly spaced paired-pits and pit membranes that hydraulically connect neighboring xylem elements. Hairpin-RNA knock-down constructs reducing PRP expression in Medicago truncatula hairy root tumors disrupted cortical and vascular patterning. Immunoblots showed that the knockdown tumors had potentially compensating increases in the non-targeted PRPs, all of which cross-react with the anti-PRP antibodies. However, PRP3 knockdown differed from knockdown of PRP1 and PRP2 in that it greatly reduced viability of hairy root tumors. We hypothesize that repetitive PRPs interact with acidic pectins to form block-copolymer gels that can play distinct roles in legume root cell walls.

     

Contributions of the surface of the root tip to the growth of Zea roots in soil

The development of the epidermal layer of roots of Zea is traced from the quiescent centre to the zone where root hairs develop. In the zone of cell division a three layered coat forms on the outside of the epidermal cells consisting of the outer epidermal walls, overlaid by a two-layered pellicle composed of a thick fibrillar inner layer of polysaccharide, and a thin fibrillar outer layer of protein. The epidermal cells divide several times in the same longitudinal file but rarely across a radius to give a new longitudinal file. Thus, the radial walls become much thicker than all but the original transverse walls, and packets of up to 32 daughter cells derived from a single initial may be distinguished. The pellicle develops during these divisions as a continuum over the outer walls of the daughter cells. It is proposed that the pellicle provides a stiffening to the forward end of the root which permits it to penetrate soil without bending. Support for this hypothesis is shown by the Zea mays mutant Ageotropic in which the pellicle is absent, the epidermal surface is disorganized, and which grows crookedly through soil. In the zone of extension growth of normal roots of two Zea species the pellicle thins and disappears. Circumferential strips of the pellicle were peeled off the young epidermal cells and could be stretched to twice their length. This deformation is partly the result of the pellicle stretching and breaking above the attachments of the radial walls. After normal thinning of the pellicle, detachment of the radial walls at their outer ends produces a corrugated surface in the proximal zone of the root tips. In dicotyledons (e.g., soybean), there is no similar pellicle, but a stiff root tip is produced by a long multi-layered root cap, the proximal portion of which covers the elongating epidermal surface.     

Growth and Differentiation of Root Endodermis in Primula acaulis Jacq.

Adventitious roots of Primula acaulis Jacq. are characterized by broad cortex and narrow stele during the primary development. Secondary thickening of roots occurs through limited cambial growth together with secondary dilatation growth of the persisting cortex. Close to the root tip, at a distance of ca. 4 mm from the apex, Casparian bands (state I of endodermal development) within endodermal cells develop synchronously. During late, asynchronous deposition of suberin lamellae (state II of endodermal development), a positional effect is clearly expressed - suberization starts in the cells opposite to the phloem sectors of the vascular cylinder at a distance of 30 – 40 mm from the root tip. The formation of secondary walls in endodermis (state III of endodermal development) correlates with the beginning of secondary growth of the root at a distance of ca. 60 mm. Endodermis is the only cortical layer of primrose, where not only cell enlargement but also renewed cell division participate in the secondary dilatation growth. The original endodermal cells additionally divide anticlinally only once. Newly-formed radial walls acquire a typical endodermal character by forming Casparian bands and deposition of suberin lamellae. A network of endodermal Casparian bands of equal density develops during the root thickening by the tangential expansion of cells and by the formation of new radial walls with characteristic wall modifications. These data are important since little attention has been paid up till now to the density of endodermal network as a generally significant structural and functional trait of the root. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mycorrhizal infection and ageing affect element localization in short roots of Norway spruce [Picea abies (L) Karst.]

Norway spruce [Picea abies (L.) Karst.] seedlings, nonmycorrhizal of mycorrhizal with Laccaria laccata or Paxillus involutus were grown in a quartz sand-nutrient solution system for 6 months and then treated with 5 M Pb for 4 days. Element contents of cortex cell wall of young, medium and old short roots were determined by X-ray microanalysis of longitudinal thin sections. The Pb content was influenced neither by age nor by the distance from the root tip (up to 1.7 mm) but was significantly lower in the P. involutus mycorrhizae than in the L. laccata mycorrhizae or in nonmycorrhizal short roots. In the P. involutus mycorrhizae, the P content of the cortex cell walls was twice as high in young mycorrhizae than in old mycorrhizae. In the nonmycorrhizal short roots and the L. laccata mycorrhizae, P content was influenced neither by age nor by distance from the root tip. The Ca and Fe contents of the cortex cell walls increased with age in the nonmycorrhizal short roots and the mycorrhizae. It is concluded that the element content of the cortex cell walls of short roots is strongly influenced by age, while the distance from the root tip seems to be of minor importance.     

On the evidence of a diffusion barrier in the outer cortex apoplast of cress-roots (Lepidium sativum), demonstrated by analytical electron microscopy

To mark the apoplastic pathway of ions in the root of the dicotyledonous plant Lepidium sativum we used the heavy element lanthanum, which can be identified by analytical electron microscopy (EELS and ESI). In the front root tip, the primary walls of all meristematic cells contained lanthanum. 10-15 mm behind the root apex, lanthanum was found in the cortex cell walls up to the endodermis, but not in the stele. 20-25 mm from the tip, lanthanum was accumulated in the radial cell walls of the hypodermis, which, however, is not a complete diffusion barrier for ions, so that traces of lanthanum also were found in the cortex cell walls up to the endodermis. This study provides evidence for the presence of two apolastic diffusion barriers in the region of highest water uptake in cress roots.     

In vitro loss of nucleic acids from cells of aseptically cultured excised pea roots

Summary Analysis of pea root tips taken from attached seedling roots and excised roots cultured in vitro has revealed major differences in cell constituents. The cells of cultured roots have only 40% and 13% of the protein and amino acid content of attached root cells. The nucleic acid content of cultured root cells was shown to be only 20% and 27% of the RNA and DNA respectively found in attached roots. It is suggested that there is excess nucleic acid in whole plant tissues above that required for transfer of genetic information necessary for normal growth and differentiation of root cells.     

Properties of lead deposits in cell walls of radish (Raphanus sativus) roots

Various mechanisms are involved in detoxification of heavy metals such as lead (Pb) in plant cells. Most of the Pb taken up by plants accumulates in their roots. However, the detailed properties of Pb complexes in roots remain unclear. We have investigated the properties of Pb deposits in root cell walls of radish (Raphanus sativus L.) seedlings grown on glass beads bed containing Pb pellets, which are the source of Pb-contamination in shooting range soils. Pb deposits were tightly bound to cell walls. Cell wall fragments containing about 50,000 ppm Pb were prepared from the roots. After extracting Pb from the cell wall fragments using HCl, Pb ions were recombined with the Pb-extracted cell wall fragments in a solution containing Pb acetate. When the cell wall fragments were treated with pectinase (E.C. 3.2.1.15) and were chemically modified with 1-ethyl-3-dimethylamino-propylcarboimide, the Pb-rebinding ability of the treated cell wall fragments decreased. When acid-treated cell wall fragments were incubated in a solution containing Pb²⁺ and excess amounts of a chelating agent, Pb recombined with the cell wall fragments were measured to estimate the affinity between Pb²⁺ and the cell wall fragments. Our data show that Pb²⁺ binds to carboxyl groups of cell walls. The source of the carboxyl groups is suggested to be pectic compounds. A stability constant of the Pb-cell wall complex was estimated to be about 10⁸. The role of root cell walls in the mechanism underlying heavy metal tolerance was discussed.     

THE WOOL-LIKE COVERING OF THE ROOTS OF LANNEA ALATA I. THE ORIGIN AND MORPHOLOGY OF THE WOOLY COAT

Lannea alata (Engl.) Engl., Anacardiaceae, a tree or shrub of East Africa, has roots covered with dense wool-like hair. Cork cambium of the root produces a closely appressed cork from which the hairs (modified cork cells) arise. Cork cells and wooly hair are rich in sterol and carotin-oid-like compounds and have thick walls. It is suggested that the root wool plays a role in the soil-air, soil-water relations of Lannea alata and other plants.     

Ultrastructural studies of Phellinus sulphurascens infection of Douglas-fir roots and immunolocalization of host pathogenesis-related proteins

Interactions between roots of Douglas-fir (DF; Pseudotsuga menziesii) seedlings and the laminated root rot fungus Phellinus sulphurascens were investigated using scanning and transmission electron microscopy and immunogold labelling techniques. Scanning electron micrographs revealed that P. sulphurascens hyphae colonize root surfaces and initiate the penetration of root epidermal tissues by developing appressoria within 2 d postinoculation (dpi). During early colonization, intra- and intercellular fungal hyphae were detected. They efficiently disintegrate cellular components of the host including cell walls and membranes. P. sulphurascens hyphae penetrate host cell walls by forming narrow hyphal tips and a variety of haustoria-like structures which may play important roles in pathogenic interactions. Ovomucoid–WGA (wheat germ agglutinin) conjugated gold particles (10 nm) confirmed the occurrence and location of P. sulphurascens hyphae, while four specific host pathogenesis-related (PR) protein antibodies conjugated with protein A–gold complex (20 nm) showed the localization and abundance of these PR proteins in infected root tissues. A thaumatin-like protein and an endochitinase-like protein were both strongly evident and localized in host cell membranes. A DF-PR10 protein was localized in the cell walls and cytoplasm of host cells while an antimicrobial peptide occurred in host cell walls. A close association of some PR proteins with P. sulphurascens hyphae suggests their potential antifungal activities in DF roots.     

Root hair-specific EXPANSIN B genes have been selected for graminaceae root hairs

Cell differentiation ultimately relies on the regulation of cell type-specific genes. For a root hair cell to undergo morphogenesis, diverse cellular processes including cell-wall loosening must occur in a root hair cell-specific manner. Previously, we identified and characterized root hairspecific cis-elements (RHE) from the genes encoding the cell wall-loosening protein EXPANSIN A (EXPA) which functions preferentially on dicot cell walls. This study reports two root hair-specific grass EXPB genes that contain RHEs. These genes are thought to encode proteins that function more efficiently on grass cell walls. The proximal promoter regions of two orthologous EXPB genes from rice (Oryza sativa; OsEXPB5) and barley (Hordeum vulgare; HvEXPB1) included RHE motifs. These promoters could direct root hair-specific expression of green fluorescent protein (GFP) in the roots of rice and Arabidopsis (Arabidopsis thaliana). Promoter deletion analyses demonstrated that the RHE motifs are necessary for root hairspecific expression of these EXPB promoters. Phylogenetic analysis of EXP protein sequences indicated that grass EXPBs are the only orthologs to these root hair-specific EXPBs, separating dicot EXPBs to distal branches of the tree. These results suggest that RHE-containing root hair-specific EXPB genes have evolved for grass-specific cell wall modification during root hair morphogenesis.     

Plasmolysis as a Tool to Reveal Lead Localization in the Apoplast of Root Cells

In order to analyze the distribution of lead between cell walls and plasmalemma, two-day-old maize seedlings (Zea mays L.) were incubated for 24 h on a solution of lead nitrate at a concentration causing 50% inhibition of root growth (10^–5 M). Using the histochemical technique (precipitation of lead dithizonate), the distribution of lead in plasmolyzed and nonplasmolyzed cells of the root cortex was compared. This allowed us to separate the lead bound by cell walls from the lead located on the protoplast surface and in the periplasmic space. The plasmolysis was conducted prior to histochemical reaction by the incubation of seedling roots in 0.6 M sucrose solution for 30 min. The lead precipitates were located in cell walls and on the surface of protoplast. A small amount of lead was found in periplasmic space of some cells in root cortex. It is suggested that the lead is bound not only to the cell wall matrix but also to the plasmalemma.     

The Casparian Strip of Clivia miniata Reg. Roots: Isolation,Fine Structure and Chemical Nature*

The root endodermis of Clivia miniata Reg. was successfully isolated using the cell wall degrading enzymes cellulase and pectinase. The enzymes did not depolymerize those regions of the primary cell walls of anticlinal endodermal root cells where the Casparian strips were located. Since the endodermis of C. miniata roots remained in its primary developmental state over the whole root length, endodermal isolates essentially represented Casparian strips. Thus, sufficient amounts of isolated Casparian strips could be obtained to allow further detailed investigations of the isolates by microscopic, histochemical and analytical methods. Scanning electron microscopy revealed the reticular structure of the Casparian strips completely surrounding the central cylinder of the roots. Whereas in younger parts of the root only the anticlinal cell walls of the endodermis remained intact in the isolates, in older parts of the root the periclinal walls also restricted enzymatic degradation due to the deposition of lignin. Extracts of the isolates with organic solvents did not reveal any wax-like substances which might have been deposited within the cell wall forming a transport barrier, as is the case with cutin and suberin. However, several histochemical and analytical methods (elemental analysis and FTIR spectroscopy) showed that the chemical nature of the Casparian strips of C. miniata roots can definitely be a lignified cell wall. These findings are in complete agreement with studies carried out at the beginning of this century on the chemical nature of the Casparian strips of several other plant species. The implications of these results concerning apoplasmatic transport of solutes and water across Casparian strips are discussed.     

<Emphasis Type="Italic">Mycena</Emphasis> sp., a mycorrhizal fungus of the orchid <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

An endophytic fungus, F-23, was isolated from the roots of Dendrobium officinale Kimura et Migo, an endangered Chinese medicinal plant. The sequence of the ITS region indicated that the isolate belongs to the genus Mycena. After 4 months of inoculation, the root systems of D. officinale that were inoculated with F-23 fungus were much larger than the control’s root systems. We also observed that the hyphae of F-23 penetrated the epidermal cells within the host’s roots and spread from cell to cell. A large number of pelotons existed in the root cortical cells of D. officinale inoculated with F-23 fungus. Intracellular hyphae crossing through the host walls were also observed using SEM (scanning electron microscopy). In contrast, light microscopy and SEM showed that the transverse sections of the roots of control plants remained uncolonized. Therefore, the F-23 fungus can form mycorrhizal associations with the roots of its host plant, D. officinale, and enhance the growth of seedlings and roots. In brief, Mycena sp. was identified and shown to be a mycorrhizal fungus of the epiphytic orchid, D. officinale. This might be of potential use to the mass cultivation of D. officinale under artificial conditions.     

Structural characteristics of root–fungus associations in two mycoheterotrophic species, Allotropa virgata and Pleuricospora fimbriolata (Monotropoideae), from southwest Oregon, USA

All members of the Monotropoideae (Ericaceae), including the species, Allotropa virgata and Pleuricospora fimbriolata, are mycoheterotrophs dependent on associated symbiotic fungi and autotrophic plants for their carbon needs. Although the fungal symbionts have been identified for A. virgata and P. fimbriolata, structural details of the fungal–root interactions are lacking. The objective of this study was, therefore, to determine the structural features of these plant root–fungus associations. Root systems of these two species did not develop dense clusters of mycorrhizal roots typical of some monotropoid species, but rather, the underground system was composed of elongated rhizomes with first- and second-order mycorrhizal adventitious roots. Both species developed mantle features typical of monotropoid mycorrhizas, although for A. virgata, mantle development was intermittent along the length of each root. Hartig net hyphae were restricted to the host epidermal cell layer, and fungal pegs formed either along the tangential walls (P. fimbriolata) or radial walls (A. virgata) of epidermal cells. Plant-derived wall ingrowths were associated with each fungal peg, and these resembled transfer cells found in other systems. Although the diffuse nature of the roots of these two plants differs from some members in the Monotropoideae, the structural features place them along with other members of the Monotropoideae in the “monotropoid” category of mycorrhizas.     

Effects of population density on alienicolae of Aphis fabae Scop.: The expression of migratory urge among alatae in the field

Inspection of naturally or artificially infected Hevea roots showed that Forms lignosus can penetrate undamaged roots directly, but does so more readily through wounds or natural openings like lenticels, or through the bases of lateral roots and bark scales. Therefore, Pomes-infected trees should be identified by leaf symptoms rather than by uncovering and inspecting roots, as this generally leads to root injury, which facilitates fungal penetration. Initial fungal entry into host tissue appears to be by mechanical pressure alone, but deeper penetration is through the action of extracellular enzymes. The fungus remains intercellular in the cortex but is intracellular in the woody tissue. Ray cell walls are penetrated mechanically, but the xylem through pits. The time taken for various stages of infection to occur is assessed. The amount of damage done by the fungus to roots and the blocking of xylem vessels by tyloses suggest that yellowing, curling and buckling of leaves on infected trees are drought symptoms and not a reaction to fungal toxins. The host reacts to the invasion of the cortex by forming a cork cambium and to the invasion of the woody tissue by blocking individual cells with phenols and resins, which could be important when breeding disease resistant Hevea root stocks.