Planta (1982)156:45 61

P I ~ ~ 9 Springer-Verlag 1982

The rhizosphere in Zea: new insight into its structure and development J. Vermeer and M.E. McCully* Department of Biology, Carleton University, Ottawa, Ont. KIS 5B6, Canada

Abstract. Some of the nodal roots of field-grown Z e a m a y s L. bear a persistent soil sheath along their entire length underground except for a glistening white soil-free zone which extends approximately 25 mm behind the root cap. These roots are generally unbranched. The histology of the surface and the rhizosphere of the sheathed roots has been examined by correlated light and electron microscopy. All mature peripheral tissues including root hairs, are largely intact and apparently alive where enclosed by the soil sheath. The sheath is permeated by extracellular mucilage which is histochemically distinct from the mucilage at the epidermal surface, but similar to that produced by the root cap. Isolated cells resembling those sloughed from the sides of the root cap persist in the soil sheath along the length of these roots. Fresh whole mounts of the sheath show that these detached cells may be alive and streaming vigorously even at some distance from the root cap. Rhizosphere mucilage is associated with the isolated cells.

Key words: Cells, detached - Mucilage - Rhizosphere - Root, structure and development Z e a

Introduction

The outer boundary of the root has traditionally been pictured by anatomists as clearly defined and static. In contrast, workers who have studied the structure and microbiology of the surface and adjacent rhizosphere of field-grown roots have shown an ill-defined root boundary complicated by the presence of extracellular mucilages, deteriorated surface tissues and their debris, sloughed-off root cap cells, and both invasive and non-invasive mi* To whom correspondence should be addressed

croorganisms (see, e.g., reviews by Mosse 1975 and Balandreau and Knowles 1978). Interpretation of the published micrographs is made more difficult by the formidable problems involved in faithfully preserving and in sectioning the surface regions of field-grown roots. Problems of interpretation are increased by the frequent absence of information on developmental aspects of the structures involved and about the surface characteristics of roots of the same plants grown under axenic conditions. The root which has been best characterized with respect to its surface features and their development is the primary root of axenically grown seedlings of Z e a m a y s L. The structural and biochemical aspects of mucilage secretion by the outer cells of the root cap (see, e.g., Kirby and Roberts 1971 ; Wright and Northcote 1974; Paull and Jones 1975; Wright 1975), as well as the development of the epidemal cells and their associated mucilage have been studied (Clarke et al. 1979), and the histochemistry of the mucilages of both epidermis and root cap has been comparatively investigated (Miki et al. 1980). Thus we considered corn to be the best species to use to define the root boundary and the structural contribution of the plant to the rhizosphere in field-grown material. In this paper we present the first results obtained in this continuing study. Materials and methods Plant material Zea mays L. cv. Seneca Chief (supplied by Robson Seeds Farm

Corp., Hall, N.Y., USA) was grown in the garden plot, Carleton University in the summer of 1980. The soil was clay loam of good fertility, organic matter content of 4.4% and pH 6.5. The summer was hot and fairly dry producing excellent growth conditions for the corn. Roots were sampled at weekly or bi-

0032-0935/82/0156/0045/$03.40

46 weekly intervals during the growing season so that those developed at successively higher nodes were examined. In each case the root system was severed with a spade approx. 30-50 cm below the soil surface and the u p p e r portion gently worked free and shaken lightly. These roots and attached soil were placed in a plastic bag on ice and immediately transferred to the laboratory. Most of the roots examined were nodal roots lacking emerged laterals. They were 5-30 cm long and had originated both below and above the soil surface. Except for a soil-free region at their tip, the underground portions of these roots were encased in a persistent soil sheath (Fig. l). In a few cases samples were taken of roots which were similar except that they had developed a few laterals. Roots to which soil did not remain clinging after excavation (Fig. 1) were not sampled. During the 1981 season cv. Seneca Chief, a commercial grain corn hybrid, PAG SX-111 (Carleton Farm Seed Supply, North Gower, Ont.) and three primitive corns, Chococito, Nal Tel and Chapalote (obtained from Dr. J. Harlin, Crop Evolution Laboratory, University of Illinois, Urbana, USA) were grown in a plot at the Central Experimental Farm, Ottawa. Soil conditions were comparable to those of the University plot but the season was poor for corn growth being excessively cold and wet. The root systems of these plants were excavated as described above and examined for the presence of roots with persistent soil sheaths.

Preparation of sections of fixed and embedded tissues Cross sections, 2 mm thick, of root tissues and material clinging to the surface were taken at the root tip and at 1-, 2-, 3- and 4-cm intervals proximal to it, in the transition area between the clean and the soil-sheath-bearing regions, in the mid-sheath region, and at the root base. Similar sections were cut from randomly selected regions of the more mature roots. The segments were excised in a drop of 3% (v/v) glutaraldehyde (8% stock; JB EM Services, Montreal, P.Q. Canada) in 0.025 M potassium-phosphate buffer, pH 6.8, then transferred to fresh fixative for 12 h on ice. The fixed pieces were rinsed thoroughly with a least three changes of phosphate buffer (0.05 M, pH 6.8). For histochemistry and routine light microscopy, glutaraldehyde-fixed tissue was dehydrated in a methyl cellosolve, pr 0panol, ethanol, butanol series and embedded in a glycol methacrylate (GMA) monomer mixture consisting of 95 ml G M A (Hartung Associates, Camden, N.J., USA), 5 ml propylene glycol 200, and 0.15 g 2,2'-azobis (Eastman Chemical Co., Rochester, N.Y., USA). For electron microscopy the glutaraldehyde-fixed tissue was postfixed in 2% (v/v) osmium tetroxide (4% stock - JB EM Services) in 0.025 M phosphate buffer, pH 6.8 for 1-2 h on ice, rinsed three times with 0.05 M phosphate buffer, dehydrated in an acetone series and embedded in Spurr's resin with a minimum infiltration period of three days. Sections (2-3 gm thick) of G M A embedded tissues were cut dry with a diamond knife, placed in small drops of water on glass slides then heated onto slides overnight at 40 ~ C. Purple-gold to gold coloured sections of Spurr's resin-embedded material were expanded to silver colour with xylene vapour before being picked up on uncoated grids (thinner sections disintegrated or were unstable under the electron beam because of lack of infiltration of the soil). These sections were stained with aqueous uranyl acetate and lead citrate. Thick (0.5 gin) monitor sections were dried down on slides. For each region of the root studied, sections from at least eight blocks from different roots were examined by electron microscopy. Histochemistry was done on sections of four to six blocks for each region.

J. Vermeer and M.E. McCully: The rhizosphere in Zea

Staining and other histochemical procedures a) Toluidine blue. GMA-embedded sections were stained in 0.05% (w/v) toluidine blue 0 (C.I. #52040, BDH Chemicals Ltd., Poole, England) in benzoate buffer, pH 4.4 then thoroughly rinsed with tap water and air dried. To retain maximum metachromasy the sections were breathed on and mounted in immersion oil immediately after the last traces of vapour had evaporated. Thick sections embedded in Spurr's resin were stained with 1% (w/v) toluidine blue 0 in 1% (w/v) aqueous borax. b) Aleian blue 8GS. Sections embedded in G M A were stained with 0.3% (w/v) aqueous alcian blue 8 GS (JB EM Services, Lot #71123) for 30~45 min, washed quickly in tap water, air dried and mounted in immersion oil. c) Periodic acid-Schiff's (PAS) reaction. Section embedded in G M A were reacted with saturated aqueous dinitrophenyl-hydrazine to block inherent aldehydes then treated by the PAS procedure, air dried, and mounted in immersion oil. d) Calcofluor White. Sections embedded in G M A were stained for 2 min in 0.01% (w/v) aqueous Calcofluor White M2R New (Cyanamid of Canada, Montreal, P.Q.), rinsed, air dried and mounted in immersion oil. e) Lectin localization of terminal fucose residues'. Hand sections were cut and their autofluorescence blocked with osmium tetroxide vapour prior to reaction with fluorescein isothiocyanate (FITC)-labelled Lotus purpureus lectin (Sigma Chemical Co., St. Louis, Mo., USA), as described by Vermeer and McCully (1981). With sections embedded in Spurr's resin, the resin was first removed by treatment with sodium methoxide. Sections were then incubated in the same lectin solution (Vermeer and McCully 1981) for 2 h. After incubation, the sections were thoroughly washed in potassium-buffered saline (0.1 M, pH 7.2) followed by distilled water, then air dried and mounted in immersion oil.

f ) Aniline blue. Sections embedded in G M A were mounted directly in 0.05% (w/v) aniline blue (Polysciences Inc., Warrington, Pa., U S A ; Lot #1523) in 0.067 M potassium-phosphate buffer, pH 8.5. Sections embedded in Spurr's resin were treated for 10 m with sodium methoxide, rinsed thoroughly in tap water and mounted in the aniline-blue solution.

g) Sections embedded in G M A to be examined for autofluorescence, birefringence and by phase contrast were mounted in 0.05 M KzHPO4, pH 8.6. Where a specific reference is not given for the above preparative procedures and staining methods see O'Brien and McCully (1981) for details and references. Sections of fresh axenie roots Roots were obtained from seedlings grown axenically (Clarke et al. 1979). Hand-cut sections (O'Brien and McCully 1981) were mounted in tap water and viewed with phase-contrast optics.

Whole mounts of detached cells in the rhizosphere The soil sheath around nodal roots of greenhouse-grown plants was gently scraped with a razor blade and the scrapings mounted in tap water and viewed with phase contrast optics.

J. Vermeer and M.E. McCully: The rhizosphere in Zea

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cultivars examined is composed of three distinct regions (Fig. 1): 1) a root cap to which soil clings; 2) behind this, a relatively soil-free, glistening white region, which may bear root hairs at its proximal end and which, in the roots examined, averaged 25 m m in length; and 3 ) a region which extends to the root base (or the soil surface in roots which originate above ground) which is encased in a distinct soil sheath. The sheathed region bears root hairs throughout its length. In roots examined early in the season the transition from zone 2 to zone 3 is abrupt; in late-season roots it is gradual. A soil sheath is characteristic of all young roots which do not have emerged branches. Portions of more mature roots which have branches but which still show a white tip may lack a distinct soil sheath. Many roots, particularly of older plants are buff coloured throughout, generally much branched, and do not have a soil sheath (Fig. 1). The histology of these latter roots

Microscopy and photomicrography All photomicrographs were taken through a Carl Zeiss (Oberkochen, W. Germany) Standard Universal microscope equipped with appropriate optics. An epifluorescence system was used, with exciter filter BG12, F1 beam splitter, and barrier filter 40 for autofluorescence, exciter filter UG1 and the same beam splitter and barrier filter for Calcofluor-induced fluorescence, and exciter filter BG12, barrier filter 40 and beam splitter F500 for FITC-lectin-induced fluorescence. Fluorescence images were recorded on Plus X 35 mm film (Eastman Kodak, Rochester, N.Y., USA). Other photomicrographs were taken either on this film or on Kodak (10.12.5 cm) Ektapan sheet film. A microflash unit was used for photomicrography of living cells. Thin sections were examined in a Siemens (West Berlin, Germany) 1A electron microscope at 80 kV and micrographs recorded on Kodak electron image plates.

Results Overall structure of the nodal roots. The belowground portion of young nodal roots of all the

Table 1. Comparison of staining and optical properties of cell walls and associated mucilages of the cells of the epidermis and the outer root cap, and of detached cells in the soil sheath of young nodal roots of field-grown Zea mays Property a

Epidermis

Peripheral root cap

Outer cell wall

Overlying mucilage

Root hair walls

Walls of attached cells

Autofluorescence

2~3 jade green

1~2 jade green

2~3 jade green --,yellowish green

Birefringence

3

0~ 1

Aniline blue fluorescence

Damaged cells only 2

Calcofluor fluorescence

Walls of detached cells

Detached cells in soil sheath Mucilage b

Walls

Mucilage

3 yellowish- 1-~3 green yellowishgreen

2~1 yellowishgreen

1-~2 green~ yellowishgreen

0--.1 bright green

3

3

2

2-1

0

0

0

0 (except at tip)

pit fields 3

pit fields 3

0

pit fields 3

0

1

3

2

3

2

3~ 1

3

0~ 1

PAS reaction

4

4

3

4

2

3~1

2

1~ 2

Toluidine blue staining

blue~ blue-green 3

blue~ blue-green 1

blue-green 3

bluish mauve

bluish mauve

mauve--+ pink

bluish mauve~ mauve

pink--* mauve

Alcian blue staining

0~1

0

0~1

3

2

3~2

2~3

2--*3

Fucose-specific lectin binding

1

0~

0

1

1

1

1

1

a Except for the root-cap mucilage, the ranges shown for each characteristic indicate variation encountered among different roots. A scale of 0-4 indicates relative intensities of staining, fluorescence or birefringence as determined subjectively b The intensity of autofluorescence, birefringence and staining reactions of this mucilage decreases with increasing distances from the root cap proper. This is indicated by the ranges shown and may reflect loss of mucilage during processing and-or changes in components present ~ A thin outer layer of epidermal mucilage which is not clearly distinguished by other procedures, is distinct in the apical portions of the root due to its strong lectin affinity. The thick inner layer of this mucilage is negative but where this layer thins in the extension zone of the root the whole outer surface appears positive. In older regions there is usually no lectin positivity associated with the epidermis

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was not examined. Following are our findings of the histological and histochemical characteristics of the intact tissues, detached cells and associated mucilages of the root-soil interface regions of young nodal roots, as well as characteristics of the surface of above-ground portions of such roots. The epidermis, the mucilage which tightly adheres to portions of its surface, and detached cells and their associated mucilage which are beyond the epidermal surface in the soil sheath of roots which have originated below the soil. Where a well-developed soil sheath is present it effectively doubles the root diameter (Fig. 2) and even after repeated washings (as also occur in the fixation and embedding procedures) some of this material persists (Fig. 3). The epidermis is intact, and the cells, including numerous, long root hairs are apparently alive throughout the sheath zone. The outer surface of the epidermis proper is covered by a firm mucilage layer which is uniform in thickness and clearly observed by phase contrast optics (Fig. 4). This layer does not extend beyond the base of each root hair (Fig. 4). At the most, only weak birefringence is detected in the epidermal mucilage (Fig. 5). This mucilage is intensely fluorescent in the presence of Calcofluor (Fig. 6), strongly PAS-positive (Fig. 8), stained blue-green by toluidine blue (Figs. 9, 11, 12) and not stained by alcian blue (Fig. 10). (These staining reactions are those of the thick inner layer of the epidermal mucilage. A very thin outer layer is sometimes present but it is too

J. Vermeer and M.E. McCully: The rhizosphere in Zea

narrow for its staining reactions to be distinguished reliably from 'optical' staining). The walls of the root hairs are phase light, intensely birefringent (Fig. 5), strongly stained by Calcofluor (Fig. 6) and strongly PAS positive (Fig. 8), stained dark blue-green with toluidine blue (Fig. 9) but largely not stained by alcian blue. These properties of root hair walls are closely similar to those of the walls (as opposed to mucilage) of the epidermal cells proper (Table 1). Among the sectioned profiles of root hairs in the soil sheath there are also many sections of large, isolated cells, apparently of higher-plant origin but detached from the root. These cells appear to be elongated, but because of the difficulties of sectioning the rhizosphere region, we could not determine their length. They are approx. 10 gm in diameter. Many of the detached cells show clear nuclei (Figs. 9, 11) a large central vacuole and peripheral cytoplasm. These cells can be distinguished from root hairs and epidermal cells proper by their wall characteristics (Table 1). Their walls are phase light (Fig. 4), only weakly birefringent (Fig. 5), and lightly stained by Calcofluor (Fig. 6). These walls resemble those of root hairs by giving strong PAS-positivity (Fig. 8), but unlike the latter walls, they are never stained green, but rather dark pink to bluish-mauve, by toluidine blue (Figs. 9, 11, 12) and they are stained dark blue by alcian blue (Fig. 10). The walls of root hairs, epidermal cells and the isolated cells are equally autofluorescent, though slight variation in fluorescence colour may occur (see Table 1).

Fig. 1. Root system of a three-week-old, field-grown plant of Zea mays cv. Seneca Chief gently removed from the soil. The tips have been accidently severed from the primary adventitious roots which have arisen from the mesocotyl, and from the first-order nodal roots and only mature regions of these roots, completely encased in a soil sheath remain. The two intact, second-order nodal roots are clearly divided into a soil-free region (small arrows) and a soil-encased region (large arrows). The root cap (open arrows) also binds soil. The insets show typical examples of sheathed (left) and unsheathed (right) roots, here seen in partially excavated field-grown root systems (soil removed gently by hand) of 6.5-week-old plants of the primitive cultivars (Chocoeito, left; Cha palote, right). Main figure printed from a colour transparency. Main figure x 0.8; insets x 0.47; bars = 1 cm. All material shown in the following figures is from roots of cultivar Seneca Chief. Plants were field-grown except those used for the preparations shown in Figs. 37 39. Colours referred to in reference to the black and white photomicrographs were clearly distinguished in the original preparations and have been recorded on colour transparencies. Fig. 2. Hand-cut cross-section showing part of the soil sheath of a nodal root. The sheath width (between arrows) is approximately equal to the radius of the root (R). The section is mounted in immersion oil and photographed through a 63-ram Luminar lens. Micrograph printed from a colour transparency, x 28; bar =0.5 mm Fig. 3. Hand-cut cross-section similar to that of Fig. 2 but after extensive washing. Some of the soil sheath still adheres to the surface and approximates the situation following fixation, dehydration and embedment. The young lateral root (LR) lacks a soil sheath. Photographed through a 63-ram Luminar lens and printed from a colour transparency, x 28; bar=0.5 ram, Figs. 4, 5. Cross sections of nodal roots (originated below ground level) in the sheath-bearing region (distance from the root tip ranges from 2.5 to 15 cm). Fixed tissue embedded in glycol methacrylate. The epidermis (Ep) is intact, bears living root hairs (H), and is covered with a thin layer of epidermal mucilage (closed arrows). Living, detached cells (asterisks) lie in the rhizosphere. Some soil (S) has remained in situ during processing, x 420; bars = 20 gm. Fig. 4, A relatively large amount of soil (S) has remained on this root and survived sectioning. Two detached cells (asterisks) and two root hairs (H) have been sectioned. The epidermal mucilage does not extend past the base (open arrow) of the hair roots. Phase contrast microscopy. Fig. 5. Same section as in Fig. 4 viewed through crossed polarizers. The soil sheath contains strongly birefringent quartz particles. Walls of sloughed cells are only weakly birefringent compared to walls of epidermal ceils proper and root hairs and to the epidermal mucilage

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J. Vermeer and M.E. McCully: The rhizosphere in Zea

The isolated cells are frequently embedded in a matrix which may be clumped (Fig. 7) and which is weakly Calcofhior-positive (Fig. 6), strongly PAS-positive (Fig. 8) and intensely metachromatic (pink) with toluidine blue (Fig. 9). This matrix is most clearly distinguished from the epidermal mucilage (Figs. 10, 11) by its metachromasy (epidermal mucilage is never pink) and its strong blue staining by alcian blue (epidermal mucilage is unstained). Isolated cells are always present in regions where an extensive soil sheath persists (Fig. 12). The soil itself is stained intensely pink (with a few blue-green areas) by toluidine blue; thus it is difficult to distinguish from the matrix material associated with the isolated cells in sections stained with this dye. However, the soil is unstained by the PAS procedure and only weakly stained by alcian blue, so that in sections stained by either of these procedures (Figs. 8,11) the soil clumps are surrounded by material with staining properties similar to those of the matrix.

ularly those one or two rows below the surface in the mid-flank region (Fig. 14) appear to be actively involved in synthesis and secretion of the root-cap mucilage (as is well known for similar cells in the caps of axenically grown main roots. of corn seedlings, see e.g., Morr6 et al. 1967; Paull and Jones 1975). The mucilage, which persists even around the detached cells (Fig. 13) in the most proximal regions of the cap, and the walls of both attached and detached peripheral cap cells are stained strongly pink by toluidine blue (Fig. 13). Both the walls of still-attached cap cells and the mucilage are PAS-positive (Fig. 14); the walls are strongly fluorescent in the presence of Calcofluor, though the fluorescence of the mucilage is frequently low. Both the walls and the mucilage are strongly stained by alcian blue and are birefringent and autofluorescent. Walls of detached cap cells which lie higher back along the cap flank have similar staining and optical properties, but these are generally less pronounced than in walls of stillattached cells (see Table 1).

Outer root-cap cells of in-soil roots and the mucilage associated with them. Sections through the cap of young nodal roots growing within the soil show a thin, but relatively continuous layer of soil particles which remain clinging to the outer surface of the root-cap mucilage during fixation and embedding (Figs. 13, 14). The surface regions of the root tips are morphologically and histochemically similar to those of axenically grown roots of a variety of grasses (Miki et al. 1980). Large, elongated cells (diameters in the order of 10 gm, lengths up to 40 gm) make up the outer layers of the flank of the root cap (Figs. 13, 14). Surface cells and partic-

The surface of the unsheathed portion of young insoil roots. The clean portion of the root surface (Fig. 1) more or less coincides with that region in which root elongation would be expected to be occurring. At its most distal end, this surface overlies columnar epidermal cells which are actively dividing in an anticlinal plane (Fig. 16), while at its proximal end the epidermal cells have elongated to a tabular shape (insets Figs. 15, 16) and may bear extended root hairs. The firm, thick (approx. 15 ~un) layer of mucilage which lies over the columnar epidermal cells (Figs. 15, 16) has similar staining reactions and

Figs. 6-12. Material and preparation similar to that for Figs. 4 and 5. B a r s - 20 gin. Ep, epidermis; H, root hair; asterisks, detached cells. Fig. 6. Section stained with Calcofluor White. The fluorescence induced in the epidermal mucilage is much stronger than that in the walls of the detached cells. The rhizosphere mucilage surrounding these latter cells has little affinity for the fluorochrome. Fluorescence optics, x 450. Fig. 7. The clumped nature of the rhizosphere mucilage associated with the numerous detached cells is apparent. Soil particles adhere to the mucilage (upper right and lower left of micrograph). Phase contrast optics, x 585. Fig. 8. Section treated by the periodic-acid-Schiff's (PAS) reaction. The rhizosphere mucilage (arrows) and the walls of the detached cells are moderately positive compared to the strongly positive epidermal mucilage. The soil is itself PAS-negative but contains traces of mucilage which is stained. Microorganisms with PAS-positive walls are frequently embedded in the latter mucilage (upper left of micrograph), x 315. Fig. 9. Section stained with toluidine blue. The epidermal mucilage (large arrows) is a faint green, in sharp contrast to the sloughed cell walls and the associated rhizosphere mucilage which are dark pink to bluish mauve. (The staining reaction of the epidermal mucilage referred to here and with alcian blue, Fig. 10, is that of the thick inner layer only; see text). Wails of epidermal cells and root hairs are blue to blue-green. The soil bound to the rhizosphere mucilage is intensely metachromatic (deep pink). Note the nucleus (n) in one of the sloughed cells, x 620. Fig. 10. Section stained with alcian blue 8GS. The epidermal mucilage (closed arrow) is unstained. Walls of the sloughed cells and, in this case, the small amount of surrounding rhizosphere mucilage (open arrows), are blue. The soil (5) is essentially unstained but is bound within a faint blue matrix. A portion of a nucleus (n) is just visible in one of the sloughed cells, x 770. Fig. 11. Section adjacent to that shown in Fig. 10, stained with toluidine blue. Walls of the sloughed cells and associated mucilage (open arrows) are pink. Epidermal mucilage (closed arrow) is pale blue-green, walls of epidermal cells and root hairs are strongly blue to blue-green and the soil (S) is deep pink. A nucleus (n) has been sectioned in one of the sloughed cells, x 570. Fig. 12. Section through an area of root on which an extensive region of soil sheath has survived processing, stained with toluidine blue. A single sloughed cell (strong pink staining wall) is present. The soil is deep pink. Holes in the section (Sg) were left when sand grains popped out during sectioning, x 490

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optical properties (Table 1) to those already described for the thin layer of mucilage of the epidermal cells in the soil-sheath zone (Figs. 6-12). Indeed this mucilage layer thins out (inset, Figs. 15, 16) within the clean region to reach its minimal thickness at the proximal end of this region, from where it is continuous basipetally over the outer walls of intact epidermal cells (but not their root-hair extensions). In the clean region, the epidermal mucilage does not appear to bind soil particles and the occasional clumps of soil found on this surface (Figs. 15, 16 and insets) are associated with residual root-cap mucilage.

Surface features of roots which have originated above the soil. The tips of nodal roots which have not yet reached the ground level are structurally similar to those growing within the soil except that much more root cap mucilage and many more cap cells (both detached and still fastened together) persist back over the surface of the columnar epidermis (Figs. 17-19). The staining reactions and optical properties (Table 1) of the mucilages of both the root cap and the epidermal surface of these roots are the same as those of the same materials on tips growing within the soil (cf., Figs. 1719 with Figs. 13-16) and on axenically-grown main roots (Miki et al. 1980). Many of the detached cap cells of the aerial root tips retain their nuclei and cytoplasm (Figs. 17, 19) despite the relatively harsh mid-summer field environment. In older, vigorously growing nodal roots which have originated above the soil, but whose tips have subsequently grown well into the soil, the mature regions lying just above the ground are covered with mucilage with the same appearance and staining properties as root-cap mucilage. Some bacterial

J. Vermeer and M.E. McCully: The rhizosphere in Zea

colonies are embedded in this mucilage (Figs. 2022), as are also many, apparently viable, detached cells and a large number of root hairs (Fig. 20). These hairs and the associated epidermal cells also have intact nuclei (Fig. 20) and cytoplasm. As with similar roots within soil, detached cells and root hairs are easily distinguished in sections, not only by the different staining properties of their walls with toluidine blue (root hair and epidermal walls green to blue, walls of detached cells pink) but also because root-hair walls are distinctly twolayered while walls of the detached cells are homogeneous (inset, Fig. 20). The latter distinction is more obvious in electron micrographs (see below). Below ground level on the same roots there is a similar peripheral accumulation of mucilage, root hairs and detached cells around the root. Here, however, these features are interspersed with soil particles (Figs. 23-30, 32, 35, 40). The soil is frequently poorly infiltrated by the embedding resin so that much of it together with attached mucilage and other components of the sheath are lost during sectioning. Enough of these components are retained, however, so that their fine structure can be observed. The mucilage appears to be fibrillar or at least it includes components which are fibrillar following the fixation, dehydration and embedment procedures. A limited variety of microorganisms, particularly bacteria, is found in the soil-sheath region embedded in the mucilage (Figs. 23, 24). Some of these bacteria are surrounded by an electron-lucent shell (Figs. 23, 24). Some fibrillar mucilage is always associated with the soil particles (Fig. 23) and may bind large numbers of these together (Fig. 26). Similar mucilage is frequently but not consistently found close to root hairs and large aggregates of soil may be

Figs. 13-16. Longitudinal sections cut near the tips of nodal roots which have originated below soil level. Tissues have been fixed and embedded in glycol methacrylate. Bars = 20 gm. Fig. 13. Edge of root cap on the lower flank of the root meristem. Both attached and detached cap cells are present; the outer ones are embedded in root-cap mucilage (asterisk). A few soil particles cling to the surface of this mucilage. Cap cells overlie epidermal mucilage (between arrows) or columnar epidermal cells (Ep). Section stained with toluidine blue. Root-cap cell walls and mucilage, bright pink; epidermal mucilage, blue-green; soil, dark pink. x 780. Fig. 14. Edge of root cap just distal to the end of the root meristem. The section has been treated by the PAS reaction. The cap cell walls and the mucilage surrounding the outer cells are strongly positive. Mucilage accumulates within the walls (arrows) on the centrifugal side of cells just under the surface layers. The layer of clinging soil is essentially unstained but appears dark because of its high refractility, x 230. Fig. 15. Section cut at a level approx. 1 mm proximal to the tip-cap junction and stained with toluidine blue. The epidermal mucitage overlying the columnar epidermal cells is pale blue-green. The clumps of strongly metachromatic (deep pink) soil are associated with residual root~cap mucilage (light pink). The inset shows a similarly stained section from a region approx. 4 cm proximal to that of the main figure. Here the epidermal cells are tabular in shape and the overlying mucilage much thinned, though it stains the same as that of the main figure. This is a "clean" region of the root and the few isolated clumps of soil (5) present are associated with wisps of root-cap mucilage. Main figure, x 800; inset, x 490. Fig. 16. Adjacent section to that shown in Fig. 15 stained by the PAS procedure. The epidermal mucilage (arrows) is stained red. Unstained soil (S) is bound to residual light red-stained root-cap mucilage. Inset: Adjacent section to that shown in inset, Fig. 15, treated by the PAS procedure. The intense positive staining of the epidermal mucilage (arrows) is maintained. There is no other mucilage present in this soil-free region. Main figure, x 685; inset, x 270

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held by it against the hairs (Fig. 28). The same mucilage may also hold soil against the smooth surface of the epidermal mucilage layer. Both these associations appear tenuous, however, and there is no apparent continuity of the mucilage fibrils with the root surface proper which in each case is clearly distinguished from the mucilage (Figs. 25, 27). In constrast, much of the fibrillar mucilage in the sections is closely connected to the detached cells (Figs. 24, 26). The walls of these cells are wavy in section and have outer microfibrils which seem to merge with, and are indistinguishable from, those of the mucilage (Fig. 24). Walls of the detached ceils appear homogeneous and are thus easily distinguished from the two-layered walls of the root hairs (cf. Figs. 24 and 26 with Fig. 27 and inset, and Fig. 28). Most of the detached cells have intact, peripheral cytoplasm containing mitochondria, short strands of rough endoplasmic reticulum and some dictyosomes (Figs. 24-26, 34). Cells which appear to have been dead before fixation (Fig. 33) are seen infrequently.

Comparison of features of the detached cells within the soil sheath and their associated mucilage, with those of root-cap cells and their surrounding mucilage. Sections of peripheral root-cap cells sloughed along the flanks of the root tip are strikingly similar to those of the detached cells within the soil sheath region. These cap cells usually have an intact peripheral cytoplasm with a normal component of organelles (Fig. 29) including dictyosomes with flattened cisternae and small vesicles (Fig. 30). Their walls are undulating and of variable thickness and they are surrounded by fibrillar mucilage

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identical in appearance to that in the soil-sheath region (compare Figs. 29 and 24). As the root-cap mucilage accumulates between the protoplast and the walls of the peripheral cap cells, intercellular connections are maintained for some time between such cells at primary pit fields where mucilage does not accumulate (Fig. 31). Here the plasmodesmata become densely stained and are apparently occluded prior to separation of the cells (Fig. 31). Such pit areas are readily identified by their positive reaction for callose in the presence of aniline blue (inset, Fig. 31). Detached cells within the soil sheath show a similar aniline-blue reactivity in pit-like thin spots in their walls (Fig. 32) and occluded plasmodesmata are frequently seen (Figs. 33, 34). Perhaps the best characterized marker for corn root-cap cells and their secreted mucilage is the pentose sugar fucose which has been shown to be present in high quantity in cap mucilage of several varieties of corn (e.g., see Wright 1975). (Gas-liquid chromatographic analysis by Dr. Grant Reid (see Vermeer-MacLeod 1982) has confirmed the presence of this sugar in root-cap mucilage of axenically grown primary roots of our material.) Fluorescein-isothiocyanate-labelled lectins specific for terminal fucose residues bind strongly to both walls and mucilage surrounding peripheral cap cells (Rougier et al. 1979; Vermeer and McCully 1981; Vermeer-MacLeod 1982). There is a similar strong binding of the lectin to walls and mucilage surrounding the detached cells throughout the soil sheath region (Figs. 36, 40). A little lectin binds to the surface of epidermal cells at the distal end of the sheath region but none binds to root-hair

Figs. 17-20. Longitudinal sections of glycol-methacrylate embedded nodal roots initiated above the soil. Figs. 17-19 are cut through the tips of roots which had not yet penetrated the soil. Fig. 20 is cut immediately above the soil level, from a root which had penetrated the soil for at least 5 cm. Bars =20 gin. Fig. 17. Section cut 1-2 mm proximal to the level of the tip-cap junction and stained with toluidine blue. Root-cap mucilage has accumulated on the flanks of the root. It is strongly pink, contains numerous sloughed cap cells and overlies the blue-green epidermal mucilage, x 280. Fig. 18. Portion of a slightly more mature region than that shown in Fig. 17 stained with Calcofluor White. The epidermal mucilage (arrows) is intensely fluorescent; the fluorescence of the accumulated root-cap mucilage is variable but usually less than that of the epidermal mucilage. Several sloughed cells are present, x 605. Fig. 19. Section cut from a region slightly distal to that shown in Fig. 17 and stained by the PAS procedure. The lightly red-stained cap mucilage contains sloughed cells. Cap cells still attached to each other flank the epidermis, the mucilage of which is stained deep red. x 540. Fig. 20. Section stained with toluidine blue. The accumulated root-cap mucilage is bright pink. Numerous root hairs (H), a sloughed cell (asterisk) and large bacterial colonies (B) are trapped within this matrix. The outlined region, enlarged in the inset, contrasts the single-layered cap-cell wall with the double-layered root-hair wall. Main figure, x 740; inset, x 1380. Figs. 21-24. Electron micrographs of Spurr's-resin-embedded tissues from nodal roots. Bars= 1 pm. Fig. 21. Section through a portion of the mucilage sheath in a region comparable to that in Fig. 20. Bacteria (B) and fungal hyphae (F) are embedded in finely fibrillar material, x 11,650. Fig. 22. Region similar to that of Fig. 21 showing variation in bacterial morphology and a lower density of surrounding mucilage, x 15,850. Fig. 23. Section through the soil sheath of a belowground portion of a nodal root. The bacteria are embedded in a fibrillar matrix resembling that found on the above-ground root tips (Figs. 21, 22). x 12,200. Fig. 24. Section similar to that of Fig. 23 showing the fibrillar matrix associated with a sloughed cell (SC). The outer portion of the walls of these cells is characteristically wavy and fibrils appear to splay out from it. Cytoplasm of these cells is peripheral and contains a normal complement, of organelles (mitochondria (m) and a dictyosome (d) are present in this section). The inset shows some of the variety of bacteria that are present. These are frequently surrounded by an electrontranslucent shell. Main figure, x 11,250; inset, x 15,000

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56 surfaces or to epidermal cells in more mature parts of the sheathed roots (Fig. 40). The frequent presence of nuclei and organized cytoplasm in sections of detached cells in the soilsheath region suggested that these cells were alive when fixed. To check this point, actively growing sheath-bearing roots from aerial nodes of twomonth-old greenhouse-grown plants were examined. These roots reached the bottom of 46-cmhigh pots and were largely unbranched. Whole mounts of scrapings gently removed from the soil sheath of these roots just below the soil surface from regions well removed from any other root tips frequently yielded numerous detached cells similar in size and shape to those observed in sections. Some of these cells were obviously dead although their walls remained intact. Living, actively streaming cells were frequently observed (Fig. 38). These cells have large central vacuoles, a prominent nucleus, numerous mitochondria and their cytoplasm includes a canalicular, secondary vacuole system which has been described as a feature of living higher-plant cells (e.g., Mersey and McCully 1978). With phase-contrast optics the isolated cells show thin spots in their walls which appear to be remnants of primary pit fields (Fig. 38). On one occasion, an isolated cell was seen which contained clearly distinguishable condensed chromosomes (Fig. 39). Living isolated cells are always associated with clumped soil which adheres to them (Fig. 38). Even when these cells are dead they are usually surrounded by a shell of soil aggregates. The detached cells within the soil sheath resemble cells seen in hand-cut sections and smears made from the peripheral region of the tips of axenicallygrown roots (Fig. 37). In these latter roots, elongated cells which are clearly of root-cap origin persist on the surface of the columnar epidermis (Fig. 37). These cells are also vacuolated, exhibit vigorous protoplasmic streaming, have cytoplasm rich in mitochondria and in the canalicular second-

J. Vermeer and M.E. McCully:The rhizosphere in Zea ary vacuole component, and their cell walls show remains of primary pit fields.

Discussion Soil and sand sheaths which cling tightly to grass roots have been noted by a number of early workers (see Troughton 1957) and recently by Wullstein and Pratt (1981). These grasses were all desert or sand-dune species and the implication has been that the sheath develops in response to an arid environment (see Troughton 1957). Xeric growth conditions were clearly not a factor in the development of the soil sheaths which we have observed. These are best developed on roots that have an extensive soil-free region behind the root cap and which, in general, are either unbranched or with relatively few branches. It is not known for certain, but probable, that the sheathed roots are young and actively growing though they may be 60 cm or more long and still have a well-developed sheath back to the level of the soil surface. Those who have described the soil sheaths in xeric grasses have not been in agreement regarding what is holding the sheath to the root. Some have considered that the root hairs are responsible (e.g., Wullstein and Pratt 1981, and earlier workers cited by Troughton 1957), while Price (1911) and others mentioned by Troughton (1957) have concluded that the sheath is formed by the binding of soil particles in mucilage originating from the root. Supporting the latter view is Sprent's (1975) observation that the sand particles which cling to dry roots of soybean plants are not held by the root hairs per se but are stuck to the surface of the hairs, presumably by mucilage. Also, Nambiar (1976) attributes the thicker sheath found in dry regions of a root to a local increase in mucilage production. The present work indicates that in corn, while root hairs (which are present throughout the sheath zone) must strengthen the sheath, they are

Figs. 25-30. As Figs. 21-24 but all from below-groundportions of roots. Bars= 1 jxm.Fig. 25. Section through part of a sloughed cell (SC) and adjacent epidermal cell (Ep). The cytoplasm of the sloughed cell is less dense but still contains a usual organelle complement (e.g., dictyosomes(d) and rough endoplasmic reticulum (er) are seen here). Mucilage (asterisk) associated with the wavy wall of the sloughed cell also overlies the distinct mucilage layer (arrows) of the epidermis, x 8500. Fig. 26. A portion of a sloughed cell with soil (S) bound to the associated mucilage, x 20,400. Fig. 27. Tangential section through part of a living root hair (/4) showing the characteristic two-layered wall (the outlined region is enlarged in the inset). Fibrillar mucilage lies between the hair and an underlying epidermal cell. Main figure, x6,900; inset, x 11,800. Fig. 28. A portion of a degenerate root hair (H). The associated soil particles were intermeshed with fibrillar material but this has largely disappeared because of instability of the region under the electron beam. x 13,000. Fig. 29. A portion of a detached root-cap cell (asterisk) lying on the flank of a root cap. The cell is surrounded by root-cap mucilage. Compare the characteristics of the wall of the detached cell and the surrounding mucilage with those of the sloughed cells and their associated rhizosphere mucilage in the soil sheath zone (see Figs. 24-27). Note also the differencein staining characteristics of the walls of these cells and those of the root hairs (see Figs. 27, 28). x 8,000. Fig. 30. Cytoplasm containing dictyosomes in a root-cap cell comparable to that seen in Fig. 29, x 28,000

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58

probably not primarily responsible for the adhesion of the soil aggregates. The best evidence for this is the occasional presence of fully elongated hairs toward the proximal end of the clean zone which are not associated with clinging soil; any soil residues on the otherwise clean epidermal surface seem to be stuck by small deposits of mucilage left behind by the advancing root cap. The strong affinity of root-cap mucilage for soil is demonstrated by the persistence of particles on the cap surface throughout fixing and embedding procedures (Figs. 13, 14). There is little doubt that the mucilage in the soil-sheath region plays a major role in maintaining the integrity of the sheath since it is always closely associated with soil aggregates that have remained in situ (Figs. 7-11, 32, 35). Presumably only a relatively small amount of the mucilage present in the sheath is retained during processing, the rest having remained stuck to the detached soil. This is suggested by the large deposits of mucilage which remain on older parts of aerially initiated roots just above ground level (Fig. 20); such regions have little attached soil so that mucilage is not pulled away during processing. Comparison of the staining reactions of the surface features of the field-grown corn roots with toluidine blue and alcian blue (Table 1) indicates similarity between the mucilages of the soil-sheath

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region and the root cap, and a distinct difference between these and the epidermal surface mucilage and the root-hair walls. The pattern of binding of fucose-specific lectin (see also Vermeer and McCully 1981; Vermeer-MacLeod 1982) strongly supports this conclusion and the other staining reactions and optical properties summarized in Table 1 are not inconsistent with it. Support comes also from earlier work with axenically grown roots of corn and other grasses which shows similar distinctions between root-cap-derived mucilage and the epidermal surface mucilage (Clarke et al. 1979; Miki et al. 1980; Vermeer and McCully 1981). Similarity between the root-sheath mucilage and that produced by the root cap is also indicated by chemical analyses. Floyd and Ohlrogge (1970, 1971) determined the component sugars in the gelatinous mucilage accumulated on mature aerial portions of nodal roots of corn. (This mucilage is presumably equivalent to that within the soil and in the present study shows the same histochemical reactions.) Their results were similar to those obtained by others for root-cap mucilage (see Table 1, Barlow 1975; Wright 1975). The mucilage within the soil sheath is always associated with the detached cells; thus the origin of these cells is of considerable interest. They are clearly distinguished from the epidermis and root hairs, and since the latter cells are intact and large-

Figs. 31-36. Electron or optical micrographs of Spurr's-resin-embedded tissues from nodal roots. Bar in Figs. 31, 33-34 = 1 ~tm, in the other figures and inset to Fig. 31 =20 gm. Fig. 31. Portions of two peripheral root-cap cells on the flank of a root tip. These cells are still attached to each other. The protoplast of the outermost cell (right of micrograph) is degenerate but still joined to that of the apparently living inner cell by densely stained plasmodesmata in primary pit fields. The living cell has an accumulation of loosely packed fibrillar material (asterisk), presumably root cap mucilage, between the plasma membrane and the celt wall. The inset shows longitudinal sections of similar cells mounted in aniline blue solution and viewed in a fluorescence microscope. Pit fields (arrows) show the characteristic fluorescence of callose. There is a similar fluorescence at the tips of the elongated, outermost cells. Main figure, x 13,350; inset, x 175. Fig. 32. Longitudinal section through the soil-sheath region of a nodal root showing sloughed cells (asterisks). The section has been mounted in aniline-blue solution and viewed with fluorescence optics. Fluorescing spots (arrows) in the walls of the sloughed cells appear to correspond to those in pit fields of peripheral root-cap cells (Fig. 31). The apparent fluorescence of the soil is due to fluorochrome trapped under the loosened portion of the section, x 560. Figs. 33, 34. Remains of plasmodesmata (arrows) in the walls of a degenerate (Fig. 33) and a living (Fig. 34) sloughed cell in a region of soil sheath comparable to that shown in Fig. 32. Such wall areas are presumed to be the sites of the aniline-blue-induced fluorescence (Fig. 32). Fig. 33, x 13,350; Fig. 34, x 13,650. Fig. 35. Cross section in the soil-sheath region of a mature nodal root showing sections through numerous sloughed cells (asterisks) and associated mucilage. The outlined region is shown in Fig. 36. Spurr's-resin-embedded material, stained with toluidine blue. x 300. Fig. 36. The section shown in Fig. 35 was incubated in FITC-fucose-specific lectin following removal of the embedding resin. Lectin binding occurs in the walls and mucilage surrounding sloughed cells. The area shown is that outlined in Fig. 35. Fluorescence optics, x 1,700. Fig. 37. Longitudinal hand-cut section mounted in tap water of a fresh, axenically-grown root showing living, detached root-cap cells clinging to the surface of the epidermis. These cap cells exhibited vigorous protoplasmic streaming. The region shown is approx. 1.8 mm proximal to the level of the tip-cap junction. Here the columnar epidermal cells are just beginning to elongate to the tabular shape. Phase contrast optics, x 400. Figs. 38, 39. Whole mounts in tap water of material gently scraped from the soil-sheath region of unbranched nodal roots of greenhouse-grown plants. Phase-contrast optics, x 800. Fig. 38. A sloughed cell which is vigorously streaming. The nucleus is clearly visible and the cytoplasm contains numerous mitochondria and an actively moving secondary vacuole component (small arrows). Regions where walls are thinner (presumed to be primary pit fields) can just be distinguished (large arrows). Soil particles (S) typically cling strongly to such isolated cells. Fig. 39. Condensed chromosomes (arrows) in a cell similar to that shown in Fig. 38. Fig. 40. Hand-cut section across the soil sheath of a fresh nodal root. The section was treated with OsO4 vapour to quench autofluorescence before reaction with FITC-fucose-specific lectin. There is strong fluorescence in the walls and the mucilage surrounding the sloughed cells (asterisks). Fluorescence optics, x 320

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60 ly alive in the roots examined, the only possible origin of the detached cells seems to be the root cap. All the fine-structural and histochemical evidence presented here supports such an origin. As far as we know there is no report of the survival and growth of isolated vegetative cell types of higher plants under field conditions. (Because the growth rates of the roots examined was not known it is not possible to estimate with any accuracy the age of the detached cells but they may be located back from the tip a distance of at least several days growth). Nutritionally there is probably no barrier to such survival in the rhizosphere. It has been established that large amounts of fixed carbon are secreted from the surface of plant roots, particularly those of the grasses (Russell 1977). Much of the secreted carbon is in the form of sugar but a wide range of amino, acids, organic acids, vitamins and even auxins are either released from the roots or synthesized by microorganisms in the root environment (e.g., see Rovira 1969; Scott 1972, Russell 1977; Hale and Moore 1978). In this regard, Brown et al. (1950) have shown that exudates from both corn and sorghum roots contain a factor which at low concentration stimulates the growth of isolated corn-root segments. The absence of a soil sheath on the white tips of the roots is puzzling. At present we can only suggest that because these regions are almost certainly elongating rapidly, sloughed cap cells and their surrounding mucilage do not get the opportunity to bind the surrounding soil to the expanding surface. There is also the possibility that the epidermal mucilage undergoes a change with age which facilitates the binding, or that exudates from more mature regions are involved in the adhesion of the mucilage, the detached cells and the soil. At present it is also impossible to determine whether the rhizosphere mucilage associated with the sloughed cells is entirely produced at or near the root cap and left behind by the growing tip, or if some is subsequently secreted into the sheath by the isolated cells. This point is now being investigated. High-resolution study of the structure of the rhizosphere dates from the pioneering work of Jenny and Grossenbacher (1963) with barley, and includes a number of subsequent studies of the rhizosphere of other grasses, particularly wheat (see review by Oades 1978 and references therein; Foster and Rovira 1973; Old and Nicholson 1975; Leppard and Ramamoorthy 1975; Foster 1981). All of these studies have shown fibrillar, presumably mucilaginous, material at the root-soil interface and within the rhizosphere.

J. Vermeerand M.E. McCully:The rhizospherein Zea Almost all of the transmission electron micrographs presented in these papers show apparently dead and frequently collapsed or distorted epidermal and occasionally also cortical cells (exceptional are the early micrographs of Jenny and Grossenbacher 1963 which give clear evidence of living epidermal cells adjacent to the rhizosphere mucilage). It is, therefore, difficult to relate most of these previous findings to the situation in the sheathed roots of Z e a described here, since the latter roots have an intact, apparently largely still living epidermis with root hairs throughout their length within the soil. Possibly the earlier observations have been on older, unsheathed roots which we have found in preliminary studies with corn to also have some dead surface tissue (Vermeer-MacLeod 1982). It is also possible that the sheathed roots in other grasses may differ from those of corn. Of the workers who have to date examined the fine structure of the rhizosphere only Greaves and Darbyshire (1972) have used Z e a and they do not report that its rhizosphere differs from that of the other grasses studied, though they do not distinguish sheathed and unsheathed roots. None of the previous studies have reported the free-living root-cap cells in the rhizosphere. While this again may be due to a difference between Z e a and the other grasses examined, it is more likely that, in most cases, the roots which were sectioned were not at a growth stage at which an active population of detached cells was still present. Recently Foster (1981) has interpreted his micrographs and also drawn on the interpretations of Leppard (Leppard 1974; Leppard and Ramamoothy 1975) to develop the hypothesis that much of the rhizosphere mucilage of wheat is derived from the extracellular material on the soil-facing surface of the epidermal cells. Foster finds this surface material to be composed initially of two easily distinguished main layers, a largely fibrillar inner layer and a more amorphous outer layer. These two layers clearly correspond respectively to the outer epidermal wall and the epidermal mucilage which we observe (e.g., compare Fig. 2, plate 1, Foster 1981 with Figs. 4 and 5, 9, 20). Our preliminary observations of unsheathed roots of Z e a (Vermeer-MacLeod 1982) suggest that the epidermal mucilage which persists along the length of these roots may indeed undergo some loosening which is coincident with changes in histochemical properties. This changed surface material is, however, not associated with a persistent soil sheath and is distinct from the predominant mucilage present within such a sheath. The results of the present study are indicative

J. Vermeer and M.E. McCully: The rhizosphere in Zea

of considerable heterogeneity in surface characteristics that probably exists between different roots of an individual plant under field conditions. This heterogeneity is compounded by other variations which result from the developmental sequence of surface tissues along an individual root. The finding that free-living root-cap cells and root-cap-type mucilage may both persist for some time in the rhizosphere of young roots adds a further complexity to the definition of a given root-soil boundary. It must, therefore, be recognized that the structure (perhaps also the function) of the rhizosphere may also differ locally depending upon the nature of the root tissues associated with it. Thus the developmental aspects of both the root boundary and the associated rhizosphere should be considered in all studies of root-soil interactions. This work was supported by a Natural Sciences and Engineering Research Council of Canada operating grant to M.E. McC. and a postgraduate scholarship to J.V. We would like to thank Dr. J. Harlin, University of Illinois for the seeds of the primitive corns and N. Ives for the two insets in Fig. 1.

References Balandreau, J., Knowles, R. (1978) The rhizosphere. In: Interactions between non-pathogenic soil microorganisms and plants, pp. 243-268, Dommerques, Y.R., Krupa, S.V., eds. Elsevier, Amsterdam Oxford New York Barlow, P.W. (1975) The root cap. In: The development and function of roots, pp. 21 54, Torrey, J.G., Clarkson, D.T., eds. Academic Press, London New York San Francisco Brown, R., Robinson, E., Johnson, A.W. (1950) The effects of D-xyloketose and certain root exudates in extension growth. Proc. R. Soc. London, Ser. B 136, 577 591 Clarke, K.J., McCnlly, M.E., Miki, N.K. (1979) A developmental study of the epidermis of young roots of Zea mays L. Protoplasma 98, 283-309 Floyd, K.A., Ohlrogge, A.J. (1970) Gel formation on nodal root surfaces of Zea mays. I. Investigation of the gels's composition. Plant Soil 33, 331-343 Floyd, K.A., Ohlrogge, A.J. (1971) Gel formation on nodal root surfaces of Zea mays. II. Some observations relevant to understanding its action at the root-soil interface. Plant Soil 34, 595 606 Foster, R.G. (1981) The ultrastructure and histochemistry of the rhizosphere. New Phytol. 89, 263-273 Foster, R.C., Rovira, A.D. (1973) The rhizosphere of wheat roots studied by electron microscopy of ultra-thin sections. Bull. Ecol. Res. Committ. (Stockholm) 17, 93-102 Greaves, M.P., Darbyshire, J.F. (1972) The ultrastructure of the mucilaginous layer of plant roots. Soil Biol. Biochem. 4, 443449 Hale, M.G., Moore, L.D. (1978) Factors affecting root exudation 2. 1970-1978. Adv. Agron. 31, 93-124 Jenny, H., Grossenbacher, K. (1963) Root-soil boundary zones as seen in the electron microscope. Proc. Soil Sci. Soc. Am. 27, 273 277

61 Kirby, E.G., Roberts, R.M. (1971) The localized incorporation of aH-L-fucose into cell wall polysaccharides of the cap and epidermis of corn roots. Planta 99, 211-221 Leppard, G.G. (1974) Rhizosphere fibrils in wheat: demonstration and derivation. Science 185, 1066 1067 Leppard, G.G., Ramamoorthy, S. (1975) The aggregation of wheat rhizosphere fibrils and the accumulation of soil bound cations. Can. J. Bot. 53, 1729 1735 Mersey, B., McCully, M.E. (1978) Monitoring the course of fixation of plant cells. J. Microsc. (Oxford) 114, 49-76 Miki, N.K., Clarke, K.J., McCully, M.E. (1980) A histological and histochemical comparison of the mucilages on the root tips of several grasses. Can. J. Bot. 58, 2581-2593 Morr+, D.J., Jones, D.D., Mollenhauer, H.H. (1967) Golgi apparatus mediated polysaccharide secretion by outer root cap cells of Zea mays. I. Kinetics and secretory pathway. Planta 74, 286-301 Mosse, B. (1975) A microbiologist's view of root anatomy. In: Soil microbiology, pp. 39-66, Walker, N., ed. Butterworth, London Boston Nambiar, E.K.S. (1976) Uptake of Zn 65 from dry soil by plants. Plant Soil 44, 267-271 Oades, J.M. (1978) Mucilages at the root surface. J. Soil Sci. 29, 1-16 O'Brien, T.P., McCully, M.E. (1981) The study of plant structure. Principles and selected methods. Termarcarphi Publ., Melbourne Old, K.M., Nicolson, T.H. (1975) Electron microscopical studies of the microflora of roots of sand dune grasses. New Phytol. 74, 51-58 Paul1, R.E., Jones, R.L. (1975) Studies on the secretion of maize root cap slime. II. Localization of slime production. Plant Physiol. 56, 307-312 Price, R. (1911) The roots of some North African desertgrasses. New Phytol. 10, 328-339 Rougier, M., Kieda, C., Monsigny, M. (1979) Use of lectin to detect the sugar components of maize root cap slime. J. Histochem. Cytochem. 27, 878-881 Rovira, A.D. (1969) Plant root exudates. Bot. Rev. 35, 35-57 Russell, R.S. (1977) Plant root systems. McGraw-Hill, London Scott, T.K. (1972) Auxins and roots. Annu. Rev. Plant Physiol. 23, 235 258 Sprent, J.I. (1975) Adherence of sand particles to soybean roots under water stress. New Phytol. 74, 461~463 Troughton, A. (1957) The underground organs of herbage grasses. England Commonwealth Agricultural Bureau, Farnham Royal, Bucks. Vermeer, J., McCully, M.E. (1981) Fucose in the surface deposits of axenic and field grown roots of Zea mays L. Protoplasma 109, 233-248 Vermeer-MacLeod, J. (1982) Histochemical, histological and structural study of field grown corn roots. M. Sc. thesis. Carleton University Wright, K. (1975) Polysaccharides of root-cap slime from five maize varieties. Phytochemistry 14, 759-763 Wright, K., Northcote, D.H. (1974) The relationship of root cap slimes to pectin. Biochem. J. 139, 525-534 Wullstein, L.H., Pratt, S.A. (1981) Scanning electron microscopy of rhizosheaths of Oryzopsis hymenoides. Am. J. Bot. 68, 408 419

Received 18 February; accepted 18 June 1982