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Author for correspondence: Alexander Lux Tel: +421 2 60296457 Fax: +421 2 65429064 Email: firstname.lastname@example.org
• The structure and development of the cortical layers, especially the endodermis and exodermis, and changes in the cortex caused by the secondary growth of vascular tissues are described in the adventitious roots of gentian (Gentiana asclepiadea).
• Sections along the whole axis of the soil-grown roots were observed using light microscopy; fluorescence microscopy was used to determine developmental stages of the endodermis and exodermis.
• Both endodermis and exodermis develop in three stages: Casparian band formation, suberin lamellae deposition and secondary thickening of walls. After the onset of cambial activity (20 mm from apex) cortical cells expand tangentially and subdivision of individual cells starts between 20 mm and 60 mm from apex. Highly differentiated endodermal cells are divided by 0–19 new anticlinal walls, exodermal cells by 0–3 and parenchymatous mid-cortex by 0–1.
• The additional anticlinal cell walls of the endodermis and exodermis possess neither Casparian bands nor suberin lamellae. Suberin lamellae remain continuous on the surface of extended tangential walls of both layers. There is a correlation between increasing diameter of the secondary vascular tissues and the number of endodermal cells created by subdivision of the original cells.
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In some dicotyledonous species massive secondary thickening results not only in an increase in the diameter of the central cylinder but also in a remarkable structural changes in the cortical layers. Root biologists are often unaware that there can be an extensive circumferential expansion of the root cortex that accommodates the increase in root diameter caused by activity in the vascular cambium. Thus, the cortex, or its parts, can persist in quite old secondarily thickened dicotyledonous roots. Changes in the structure of the root cortex induced by cambial thickening are poorly understood.
Plant roots are the organs responsible for water and ion uptake. Thus, it is crucial for the whole plant to maintain functionally intact root tissues. Typical endodermis has three typical cell wall modifications: Casparian band in the first developmental state, suberin lamella in the second and secondary wall thickening in the third (Bond, 1930; Guttenberg, 1968; Barnabas & Peterson, 1992). As result of these wall modifications, the endodermis becomes an apoplastic barrier regulating the radial flow of water and ions in younger parts of roots (Sanderson, 1983; Peterson et al., 1993; Steudle & Peterson, 1998). In the third state, development of thick secondary walls in the endodermis protects the vascular tissues in older roots parts (Luhan, 1954). Formation of Casparian bands may be the final state in some dicotyledons and monocotyledons (e.g. in aquatic plants of Nymphaeaceae; Seago, 2002). Endodermis responds to the growth of the central cylinder by cell dilation in the tangential dimension and in some species also by subdivision of the original endodermal cells (van Wisselingh, 1926; Scott, 1928; Bond, 1930; Esau, 1940; Weerdenburg & Peterson, 1984). When endodermal cells subdivide in the first developmental state, Casparian bands are formed on new cell walls (Bond, 1930; Weerdenburg & Peterson, 1984; Lux & Luxová, 2001). An exception has been observed in Primula acaulis, where Casparian bands are formed on the new walls, although subdivision of endodermal cells occurs well into the third developmental state (Lux & Luxová, 2004). The density of the endodermal network, in which adjacent Casparian bands of individual cells form a meshwork, depends on both dilation growth and the production and nature of additional cell walls in the original endodermal cells (Lux & Luxová, 2001).
Mid-cortex layers of roots can respond to thickening of the stele by changing their original arrangement, extending their tangential dimensions, continuing to divide or simply dying off (Esau, 1940; Luhan, 1954). Cell division and changes in the cortical tissues are also induced by the formation of lateral roots (Tschermak-Woess & Dolezal, 1953; Peterson & Moon, 1993).
The hypodermis despite its peripheral position can also be influenced by secondary growth of the central cylinder. Usually, it is shed after the beginning of cork cambium activity but before this, hypodermal cells can also expand or, very rarely, renew their mitotic activity (Luhan, 1954). Reaction of the hypodermis to secondary growth is very important. According to the latest observations nearly 91% of investigated species (Perumalla et al., 1990; Peterson & Perumalla, 1990) posses a hypodermis with Casparian bands and suberin lamellae, termed an exodermis. The exodermis is thus a functionally analogous structure to the endodermis and influences the crucial functions of uptake and radial transport of water and solutes (Peterson, 1989).
The aim of this study was to describe, using fluorescent staining and microscopy (Brundrett et al., 1988, 1991), the development of the endodermis and exodermis in gentian roots, to characterize the reaction of the cortical layers to the changes in the vascular tissues caused by cambial activity and to compare this with some other plants. Gentian was chosen because of the large amount of secondary growth in its central cylinder and the previously reported subdivision in the exodermis, which is unique to the Gentianaceae (Luhan, 1954; Kutschera & Sobotik, 1992).
Materials and Methods
Adventitious roots were obtained from mature plants of G. asclepiadea L. The plants were cultivated, 1 yr after collection from their natural locality in central Slovakia, in pots with garden soil and placed outdoors. Roots, 130–140 mm long (long roots) and 20–30 mm long (short roots) from a minimum five plants were cut transversely along the length of the whole root axis to compare the primary and secondary structures and to describe the development and changes in the cortical layers.
Roots were sectioned, stained and observed within 3 h of their excavation from the soil. Intact, undamaged roots were rinsed briefly in tap water, and then hand-sectioned. For observations of the Casparian bands, sections were stained by berberine and toluidine blue O (Sigma, Taufkirchen, Germany), which was substituted for aniline blue used in the original procedure by Brundrett et al. (1988). For observation of suberin lamella, sections were stained by Fluoral yellow 088, according to Brundrett et al. (1991). Sections were observed using a Nikon Optiphot II microscope under UV illumination and set of filters allowing wavelengths > 420 nm to pass. Photographs were taken with 400 ASA colour negative film.
Semithin sections of roots were cut at distances of 10, 20, 30 to 130 mm from the root tip. Root samples approximately 2 mm long were fixed in 5% glutaraldehyde in phosphate buffer (0.1 m, pH 7.0), postfixed in 2% osmium tetroxide, dehydrated in ethanol and propylene oxide, and embedded in Spurr resin embedding medium (Sigma). Sections approximately 1-µm thick were cut using LKB Nova ultramicrotome (LKB, Bromma, Sweden) and glass knives. Sections were stained with toluidine blue and basic fuchsin (Lux, 1981) and observed using an Axiolab microscope (Zeiss, Jena, Germany). Photographs were taken with 100 ASA black and white negative film.
For quantitative analysis of dilation and subdivision in the endodermis, five roots were cut at 10, 20, 30 to 130 mm from the root apex. Cross-sections were stained for 20 min with 0.05% toluidine blue to contrast the cells. Sections were observed with a Zeiss Axiolab microscope. The number of endodermal cells was counted, and diameter of the endodermis and the size of individual endodermal cells were measured at each distance by ocular micrometer. The percentage of root cross-section area occupied by secondary vascular tissues was calculated from the diameters of individual root tissues. Results were statistically evaluated by Excel 5.0 97, mean values (x) and standard errors (SE) were calculated.
The root system of G. asclepiadea consists of thick, fleshy storage roots from which fibrous adventitious roots grow out. Roots of this species typically have massive secondary growth and subdivision in both border cortical layers, endodermis and exodermis.
Primary root structure of 130–140 mm long roots (Fig. 1)
The exodermis is uniseriate, and exodermal cells underlying the rhizodermis are hexagonal and closely packed. They are wedge-shaped where they abut the rhizodermis and outermost mid-cortex cells. The mid-cortex consists of four or five cell layers of large, irregularly-shaped, radially arranged cells with numerous intercellular spaces, except where they abut the exodermis. The diameter of endodermal cells is about half the diameter of mid-cortex cells. The central cylinder is diarch or triarch.
Development of endodermis and exodermis
Casparian bands appear in both the endodermis and the exodermis at the distance of about 4 mm from the root tip. However, exodermal Casparian bands spread quickly over the whole anticlinal cell wall, while endodermal Casparian bands remain narrow (Fig. 2a). The protoxylem cells differentiate at the same distance. Suberin lamellae are laid down on the inner surface of exodermal cell walls simultaneously with Casparian bands formation (Fig. 2b). In the endodermis, widening of Casparian bands and formation of suberin lamellae is accelerated in the cells opposite the phloem poles (Fig. 2c). At a distance of 5 mm from the tip only a very thin deposition of lamellar suberin can be seen in a few endodermal cells which are situated opposite the phloem poles (Fig. 2e).
At a distance of about 10 mm from the root tip, Casparian bands of almost all the endodermal cells occupied their whole anticlinal wall (Fig. 2f). At this distance the majority of endodermal cells possess suberin lamellae; only individual cells near the xylem poles remain without it. At this distance the situation in the exodermis is very similar that at 5 mm, except that suberin lamellae fluoresce more strongly (Fig. 2g).
Both endodermal and hypodermal Casparian bands are as wide as the anticlinal walls of original cells at the distance of 30 mm from the apex (Fig. 2h). At this distance, suberin lamellae cover the inner surface of all original endodermal and exodermal walls (Fig. 2i).
In short adventitious roots, the initiation of Casparian bands and suberin lamellae starts simultaneously in the exodermis between 3 mm and 5 mm from the apex, as in long roots. The two xylem poles are differentiated and the endodermis is without a Casparian band at this distance. Narrow Casparian bands appear in the endodermis between 5 mm and 7 mm from the apex, but widening of these starts in cells opposite the phloem poles 15 mm from apex, which is farther than in long roots. Suberin lamellae start to form in a few endodermal cells opposite the phloem poles between 10 mm and 15 mm from the apex; this is also farther than in long roots (5–10 mm from the apex). At a distance of 20 mm from the apex only single endodermal cells opposite the xylem poles lack suberin lamellae.
Secondary root structure (described for long roots; Fig. 3)
Owing to massive cambial activity, which usually starts 10–30 mm from the apex, secondary vascular tissues occupy a majority of the root volume, e.g. at the distance of 60 mm from the apex it comprises 70–80% of the root cross area. Initially the endodermal cells is enlarged tangentially and the endodermal cells soon start anticlinal divisions. The original anticlinal cell wall of each endodermis cell remains due to the presence of its Casparian band. In early stages of cambial activity, the arrangement of mid-cortex cells changed from radial to alternating, thus keeping the same volume despite the increased pressure from enlargement of the secondary vascular tissues. Later, the number of mid-cortex cells is reduced; the majority die and the remainder divide by one new anticlinal wall. This process in mid-cortex partly compensates for the pressure of growing internal tissues on the exodermis. Therefore expansion and additional anticlinal division in the exodermal cells starts later than in the endodermis. In older root parts, the rhizodermis and all peripheral cortical layers are shed and persistent, redivided endodermis covers the root surface (Fig. 4).
Dilation and additional division in endodermis and exodermis
The subdivision in the endodermis usually starts between 20 mm and 40 mm from the apex, where all the endodermal cells possess suberin lamellae. The beginning of new endodermal cell wall formation evidently correlates with an increase in the circumference of the secondary vascular tissues during secondary growth. To maintain an intact endodermis, endodermal cells elongate tangentially 2–13 times and form 0–19 new walls. A different number of new cell walls develops in individual endodermal cells. These range from small original endodermal cells, which have divided into two or three cells or large cells divided into 20 new cells.
From the root apex to the base the number of endodermal cells, as well as the diameter of the endodermis, increases (Fig. 5). The amount of division and expansion varies in different segments of the root. Near to the apex, before starting subdivision, endodermal cells only expand tangentially. After the beginning of additional divisions up to a distance of 60 mm from the apex, the processes of division and endodermal cylinder diameter expansion go hand in hand. The amount of subdivision appears to be highest between 60 mm and 100 mm from apex. Between 80 mm and 110 mm from the apex, the diameter of secondary vascular tissues also increases faster, so that the average tangential dimensions of single endodermal cells remain constant. Near the base of the roots, the increase in the diameter of the endodermal cylinder outstrips the number of subdivisions.
The exodermal cells may also divide anticlinally, forming 0–3 new walls without Casparian bands (Fig. 6). Subdivision in the exodermis starts later than in the endodermis, usually between 25 mm and 60 mm from the root apex.
While Casparian bands remain in the original anticlinal walls of the original, endodermal and exodermal cells, the newly formed anticlinal walls do not produce Casparian bands. However, all of the expanded tangential walls produce suberin lamellae and cellulosic secondary walls (Figs 3 and 6) (the reaction with phloroglucinol and HCl was thus negative, ruling out the presence of lignin).
The characteristic steps of endodermal and exodermal ontogenesis along the whole root axis are summarized in Fig. 7.
Gentiana asclepiadea can be added to the list of plant species possessing an exodermis (Perumalla et al., 1990; Peterson & Perumalla, 1990), thus strengthening the case for the wide-spread occurrence of an exodermis in the roots of flowering plants. Development of the endodermis and exodermis of gentian root proceeds in three developmental stages. They enter the first stage – formation of Casparian bands – simultaneously, but the exodermis matures faster than the endodermis; suberin lamellae are formed at the time of the appearance of exodermal Casparian bands. Simultaneous formation of Casparian band and suberin lamella in endodermis was observed by Barnabas & Peterson (1992) in Allium cepa. Development of the endodermis is affected by the structure of vascular tissues. Endodermal cells situated opposite the phloem poles mature earlier than endodermal cells, which are closer to the xylem poles, as observed first by van Fleet (1942). In Tagetes erecta this effect has been reported during the formation of the pro-endodermis (young endodermis without Casparian bands) simultaneously with the last periclinal division of the innermost cortical layer (Guttenberg, 1968).
In gentian, the development of the endodermis and exodermis continues into the third stage. The development of the exodermis into the third stage appears to be quite rare in dicotyledons (Peterson, 1997); in gentian it may be related to the protective role of the exodermis in parts of the roots where the rhizodermis is already being shed. According to Peterson (see also Seago et al., 1999b), the secondary wall thickenings in the endodermis and exodermis represent mirror images. In gentian the situation was different, in both layers a thick cellulose secondary wall is formed at the outer tangential wall, particularly in the exodermis.
Previous studies reported earlier appearance of Casparian bands in the endodermis than in the exodermis (Fahn, 1990; Peterson & Enstone, 1996). More recently, suberinization in the exodermis, very close to the root tip and before suberinization in the endodermis, has been observed in some wetland plants, such as Typha and Phragmites (Seago et al., 1999b; Soukup et al., 2002), and in a terrestrial plant Camellia sinensis (Homma et al. 2000). The long roots of gentian belong somewhere between these examples, since the differentiation of both layers starts at the same distance from the root tip. In short roots, maturation of exodermis begins closer to the root apex than in the endodermis. Development of exodermis progresses in both short and long roots faster in gentian. The sequence and distance of endodermal and exodermal differentiation are probably closely related to features of a plant's environment (Seago et al., 1999a), but the distance of Casparian band formation appears to be species specific. It usually starts within a few millimetres from the apex (e.g. 10–20 mm in A. cepa L.; Barnabas & Peterson, 1992; and 0.2–0.3 mm from the apical meristem of the lateral root in Avena sativa; Clarkson & Sanderson, 1974). In fast-growing roots, Casparian bands mature farther from the apex (e.g. at 30–40 mm in A. cepa; Peterson & Perumalla, 1984).
Vascular cambium is formed close to the root apex in gentian and because of its activity, the secondary vascular tissues grow very quickly. Their expansion causes a die-off of root tissues that starts in the rhizodermis and progress toward inner cortical layers. Destruction of the cortex appears to be gradual because changes in cell arrangement in the mid-cortex from radial to alternate (Sinnot & Bloch, 1941) partly compensate for the increasing pressure of the growing central cylinder. This could be also the reason why original endodermal cells divide into many more new cells than do original exodermal cells. Gentiana asclepiadea is another member of the Gentianaceae in which subdivision of both endodermis and exodermis has been reported. In comparison with other members of this family, G. asclepiadea belongs to those with highest number of new anticlinal walls (e.g. in Gentiana pneumonanthe up to 17 new walls are formed and in Gentiana campestris up to 24 are formed; Kutschera & Sobotik, 1992). The relatively large numbers of additional anticlinal walls in endodermal and exodermal cells and the large volume of central cylinder support the ideas of Mager (1932) and Whitmore (1962a,b) that the occurrence of subdivision is a consequence of inner pressure caused by cambium activity. Our observations agree with these of Mylius (1913), who found that subdivision starts after a period of passive tangential expansion of the endodermal cells. The new walls may act as support, preventing the expanded endodermal cells from collapse, and the same may be true of the suberin lamellae and secondary cell wall material over the new tangential surface of both endodermal and exodermal cells. These new wall materials may also have an effect upon water/solute uptake or losses after the rhizodermis has been sloughed off. The occurrence of this unique set of wall modifications in the endodermis and exodermis needs to be examined for other plants that undergo secondary root growth.
To compare the changes in the cortical cells in relation to those in the stellar tissues caused by cambium activity, Štefanovic̆ová & Lux (2001) presented some parameters describing the changes in the root cortex of yarrow (Achillea collina) and ribwort plantain (Plantago lanceolata). These are compared with gentian in Table 1. It is obvious that these three species exhibit three different types of cortical changes. It is highly likely, therefore, that other types of endodermal and exodermal development await discovery. The occurrence of additional divisions and the patterns of endodermal cell expansion lead to major changes in the density of Casparian band network (Lux & Luxová, 2001). In gentian, the Casparian band network density is reduced both in the endodermis and exodermis, since the new walls lack Casparian bands and dilation of the original endodermal cells increases the distances between the anticlinal walls (with Casparian band), while the tangential walls maintain a complete suberin lamella barrier even though these layers are subsequently sloughed off. Experimental data are now required on the possible barrier functions of these unique and complex endodermal/exodermal networks.
Table 1. Subdivision and presence of Casparian bands in new anticlinal cell walls of cortical layers of Achillea collina, Gentiana asclepiadea and Plantago lanceolata
Abbreviations: naw, new anticlinal cell wall; +CB, new anticlinal wall possessing a Casparian band; –CB, new anticlinal wall without a Casparian band.
0–3 naw +CB
0–19 naw –CB
0–3 naw –CB
0–2(3) naw –CB
This work was supported by the grant from Slovak Grant Agency VEGA No.1/0100/03 and COST 837. We thank James L. Seago Jr for critical reading and reviewing the manuscript and helpful suggestions. We thank the referees for valuable comments and corrections.