General features of L. barbata
Rhizomes excavated in the field during winter have aerial culms. Perennial sandbinding roots emerge mostly in the vicinity of culms, but also from intercalary regions of the rhizome (Fig. A). These roots descend relatively straight and deeply into the sandy substrate. Except for their most distal regions, they are surrounded by a firmly attached sandsheath (Fig. B). Sandsheaths begin to form, on average, 10 ± 2 mm from the bare white tips, but the length of these bare regions is reduced in slower-growing roots harvested during earlier and later parts of the growing season. The bare, clean portions of the root surface (Fig. B) more or less coincide with the region of root elongation. Axile roots produced in earlier seasons can persist for several years.
Structure of the surface of the developing sandbinding roots
CSEM of roots cryo-fixed in the field confirmed that tips had normal root caps and a bare soil-free region overlying meristematic and elongation zones (Fig. A). Here, cells of the epidermis were in files along the roots and clearly showed that a root hair protruded from every cell (Fig. ). A few sand grains clung to the extreme tip regions (Fig. B), but on surfaces less than 10 mm proximal to tips, early development of sand binding was seen in associations of young root hairs elongating between and around a few sand grains of varying size (Fig. B).
As root hairs elongate, they recruit an increasing depth of sand particles into the sheath, increasing its thickness up to six-fold between 10 and 100 mm from the root tip. Over the same distance the ratio of sheath volume to that of the subtending root increased 14-fold (Table ). Tight enmeshment of the sand was ensured, not only by the extremely high density of hairs (approx. 5900 mm−1 root length, Table ), but also by their apparent thigmotropic response by which they track and tightly wrap around many of the grains (Fig. A, B). At 5 mm from the tip the root hairs were alive, with walls approx. 0·5 µm thick (Fig. C) but by 100 mm from the tip the walls were over 3 µm thick and the hairs were dead (Fig. D). The dead hairs persist and retain their smooth cylindrical shape even in very old sheaths which persist on the perennial roots (Fig. ). The strength and rigidity prevent easy removal of the sheath by 100 mm from the root tip where most of the sand could be removed only by scraping. In contrast, it was easy to remove the sheath from younger regions, 20–30 mm from the tip by a vigorous water wash.
Measurements of Lyginia barbata roots and their surrounding sandsheaths formed at increasing distance from the root apex
Measurements of epidermal cell length and width in roots of Lyginia barbata
Fig. 3. (A, B) Root hairs enmeshing sand grains (S) at the periphery of developing sandsheaths, approx. 10 and 75 mm, respectively, from the tip of a growing root like that in Fig. 1B. (C, D) Fractured root hairs, at approx. 5 and 100 mm, respectively, from a (more ...)
Fig. 4. (A) An oblique cut with a razor blade of a fresh approx. 3-year-old, perennial root with intact, sandsheath and persistent root hairs. Brown collapsed cortex and epidermis (arrows). (B) Sandsheath surrounded by a haustorium (arrow) of the root hemiparasite (more ...)
Dormant roots had sandsheaths enmeshed by thick-walled root hairs close to (Fig. E) or completely covering the root tip (Shane et al., 2009
Structural changes in associated epidermis and cortex during sandsheath development
The distance from the root tip at which the sequence of developmental changes occurs varied with the growth rate of individual roots, and in dormant roots was very short (Fig. 3E; cf. Shane et al., 2010
). Figure illustrates a typical developmental sequence of peripheral tissues with accompanying sandsheath development in growing roots. The youngest epidermal cells in the soil-free region close to the root tip are columnar, with a thick outer tangential wall (Fig. A), which forms a protective pellicle as in grasses (McCully and Canny, 1994
). Other walls of these cells and those of the young cortex are thin, and all cells are intact and closely packed, with only a few small intercellular spaces in the central cortex (Fig. B).
Fig. 5. (A–D) Roots frozen in the field, cryo-planed; CSEM. (E–G) Hand-cut, frozen in the cryo-microscope. (A) 2 mm from the root tip, root hairs not yet emerged. A thick pellicle (arrows) forms the epidermal (Ep) surface, cortex (Cx) intact and (more ...)
At increasing distance from the tip, cells in the central cortex progressively die and are crushed, eventually forming an aerenchymous middle cortex (Figs C–G and E). This aerenchyma was colonized frequently by actinobacteria (inset, Fig. G). Epidermal cells elongated to a more tabular shape, but remained relatively short, each cell retaining a root hair (Fig. C). Further from the root tip, cells of both the outer and the inner cortex develop into thick-walled fibres. Epidermal cells, in parallel with their root hairs, also develop thick walls which eventually appear completely coherent with the underlying outer cortical fibres (Fig. E–G), forming a strong supporting base which retains the root hairs (and their enmeshed sandsheath) in place. The inner cortical fibres also form a tightly packed inner rim of sclerenchyma (Fig. E–G). The endodermis remains distinct from this rim and is easily separated from it during specimen handling (Fig. F). The inner and outer rims are held together by remnants of the middle cortical walls (Fig. F). With root maturity, both the inner and the outer sclerotized rims become brown (Fig. A).
In dormant roots, all these developmental processes at the root surface and within the cortex have proceeded right to the apex (Fig. E).
Wall histochemistry of root hairs, epidermal and cortical tissues associated with sandsheath formation
The histochemical reactions on fresh hand-cut sections are illustrated in Fig. and are summarized in Table .
Fig. 6. Fresh, hand-cut transverse sections of roots after removal of sand grains. The endodermis (arrow) is toward the left of each micrograph. Approximate distance (mm) of each section from the root tip is indicated. (A–C) Toluidine blue stain, at 10, (more ...)
Wall staining properties of root hairs and cells of outer, middle and inner cortex in fresh sections of roots of L. barbata. Observations were made from each of 10–20 roots.
With Toluidine blue (Fig. A–C), pink to purple metachromasy colours were characteristic of the walls of root hairs and young epidermal cells in the soil-free region close to the root tip (Fig. A), indicating acidic polysaccharides. The cortical walls were blue, indicating aromatic compounds. The metachromasy disappeared by 20–30 mm from the tip, replaced by blue in the hair and epidermal walls, indicating masking of the acidic polysaccharide staining by aromatic residues. The cortex walls were strongly blue. The positive test for ferulic acid (Fig. J, K) on sections 20 mm from the root tip indicates that the Toluidine blue wall staining was, at least in part, due to ferulic acid incrusted in the walls. The epidermal walls included some dark-brown patches of endogenous pigment (Fig. B). At approx. 120 mm from the tip, the central cortex and root hair walls were more greenish blue, and dark-brown endogenous pigment masked and/or prevented any staining of walls of epidermis and narrow regions of both outer and inner cortex (Fig. C).
Rhodamine B-induced, yellow to orange fluorescence indicated that the walls of root hairs and epidermis included a lipid-rich component (probably suberin) as close to the root tip as approx. 10 mm (Fig. D) and this staining intensified by approx. 20 mm (Fig. E). Further from the tip, autofluorescence of aromatic compounds begins to obscure the orange fluorescence, with the result that its origin from a thin outer layer of the root hair walls becomes increasingly clear (Fig. F, G, and lower inset G). In other root tissues the underlying autofluorescence obscures any reaction with the fluorochrome (Fig. D–F). Endogenous pigment obscures cortical fluorescence by approx. 180 mm from the tip, but the endodermis is still strongly fluorescent (Fig. G).
Pale pink to red staining by Phloroglucol/HCl (Fig. H, I) indicated progressive lignification of root hairs, epidermis, and inner and outer cortex. Mid-cortical cells remain unlignified. Where present, the endogenous pigment was very dark red to black.
The thick walls of old root hairs were strongly birefringent, and Sudan staining revealed a thin lipid-rich outer layer (upper inset, Fig. G).
Sheath formation on root mesh
Of the 15 root mesh frames installed in the field (Fig. ), ten were colonized by a sandbinding root along its entire length. These ten root meshes included at least three from each mesh treatment. In some cases roots failed to grow, probably due to disturbance and/or drying out during installation. Roots that descended down against the upper surface of inclined meshes of 38- or 1-μm pore size constructed sandsheaths on their upper and on the lower surface of the mesh (Fig. A–D). Staining of sandsheaths on lower surfaces (Fig. C) showed that the root hairs had grown through the mesh and encircled and entrapped sand grains essentially as observed in sandsheaths formed in direct contact with roots elsewhere in the field. Sandsheaths formed on the lower surfaces of 1-μm pore size mesh overlaid by mature root regions were thinner (0·8 ± 0·2 mm, Fig. D) compared with those developed below 38-μm pore size mesh (1·3 ± 0·2 mm) which were similar to those measured on untreated roots (Table ).
The arrangement to test whether sandsheaths were formed by roots separated from soil by mesh of different pore size (see text). Scale bar = 100 mm.
Fig. 8. Sandsheath formation by roots growing in contact with mesh screens in the field (Fig. 7). (A) Sheath on the upper side of a root growing downward over a nylon screen (38-μm pores) beyond where the root tip first contacted the mesh (arrow) the (more ...)
Root hair diameter ranged from 8 to 12 µm and they easily penetrated mesh of pore size 38 µm. However fewer, but still a surprising number, managed to grow through 1-μm pore mesh (Fig. D) by displacing the nylon fibres (Fig. E).
The more rigid polycarbonate membranes (0·03-μm pores) were not penetrated by root hairs, and there was no sand binding on the lower surface. Staining of the polycarbonate mesh with Toluidine blue, by the PAS procedure or by Schiff's reagent alone after removal of the roots revealed that where a root had been in contact with the mesh, there were clear, unstained root hair ‘ghosts’ (Fig. A, B). Mesh between the ghosts stained blue/green with Toluidine blue, and light or strong pink with Schiff's reagent or the PAS reaction, respectively. These staining reactions indicated sparse production of polysaccharide and phenolic exudates by either the root and/or associated microbes, in contrast to the prolific accumulation of mucilage on a nylon membrane traversed by a maize root (Watt et al., 1993
; McCully, 1999
, fig. 2d).
Fig. 9. (A, B) Upper surface of mesh (0·03-μm pores) after 3 months in the field, along which a root grew, but no root hairs penetrated, and no sandsheath formed on the underside. After the root was removed gently, unstained root hair ‘ghosts’ (more ...)
Mesh beyond the contact region did not stain. The unstained root hair ‘ghosts’ were similar in appearance to surface patterns observed by CSEM where root hairs had developed in the field while appressed against a hard surface (cf. Fig. C with Fig. A, B).
Mesh surfaces were colonized by only a few fungal hyphae (Fig. A, B). CSEM of root-hair surfaces showed some possible bacterial colonies (Fig. D), and, occasionally, apparent exudation from the tips of young root hairs (arrowheads Fig. E).
Change in phosphorus concentrations during root maturation
Total [P] decreased markedly from the youngest to the oldest segments (Fig. ). The youngest had [P] > 1 mg g−1 d. wt. The next older segments coincided with the beginning of cortical maturation, and had, not surprisingly, 14-fold less [P] than the first segments which included the meristematic and elongating cells. Therefore, we used [P] of the second segments as a base value of 100 % for comparing total root [P] change as the cortex and epidermis matured. By segment 4, which included the region where the epidermis and cortex were dead but residual (Fig. ), [P] was 0·017 mg g−1 d. wt, a decrease of 76 % during maturation of the cortex and epidermis.
Fig. 10. Phosphorus concentrations in four regions along roots, from growing tips to a region of complete epidermal and cortical senescence. To better relate change in whole root [P] as this senescence progresses [P] was expressed as a percentage of that in the (more ...)