Chemical composition of Casparian strips isolated from Clivia miniata Reg. roots: evidence for lignin

Endodermal cell walls and xylem vessels were isolated enzymatically from Clivia miniata Reg. roots. Transmission-electron-microscopic investigation of cross-sections of intact C. miniata roots and scanning-electron-microscopic investigation of isolated endodermal cell walls indicated that the root endodermis of C. miniata is essentially in its primary state of development. Isolated Casparian strips and xylem vessels were subjected to two different degradation methods usually applied to prove the existence of lignin, namely, cupric oxide oxidation and thioacidolysis. The reaction products obtained were typical aromatic derivatives of the natural lignin precursors coniferyl and sinapyl alcohols, and, in traces, of p-coumaryl alcohol, indicating the occurrence of lignin in the polymers from both Casparian strips and xylem vessels. The qualitative chemical compositions of the polymers from the two sources were similar, whereas the quantitative compositions were different, indicating that the molecular structure of the lignin polymer in the Casparian strips was different from that in the xylem vessels. Thus, for the first time, direct chemical evidence has been obtained that Casparian strips of C. miniata roots contain lignin as a major cell wall polymer.The author is indebted to Prof. Dr. G. Krohne (Zentrale Abteilung für Elektronenmikroskopie, Universität Würzburg, Germany) and to Prof. Dr. R. Guggenheim (Labor für Rasterelektronenmikroskopie, Universität Basel, Schweiz) for offering the opportunity for transmission-electron-microscopic and low-temperature scanning-electron-microscopic investigations, respectively. Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.     

ADVENTITIOUS ROOT DEVELOPMENT FROM THE SEEDLING HYPOCOTYL OF LYCOPERSICON ESCULENTUM

Tomato seedlings five through ten days old were used for this investigation. Adventitious roots were initiated from the pericycle of the tomato hypocotyl. The position of adventitious root development was irregular in the rhizogenic hypocotyl; however, the cellular pattern of individual root development was very regular. Four layers of pericycle derivatives participated in root histogenesis and a bi- or triseriate endodermal cover was derived from the endodermis. Fluorescent microscopy showed that Casparian strips on the meristematic endodermal cell walls were not removed biochemically but were displaced around the root primordium by anticlinal divisions and cell enlargement. Casparian strips were not synthesized by endodermal cover cells. The emergent root had a typical three tiered or closed pattern of apical organization, and quiescent centers were present in all emergent roots longer than 0.5–0.6 cm.     

The development of the endoder mis andPhi layer of apple roots

Summary Suberin lamellae and a tertiary cellulose wall in endodermal cells are deposited much closer to the tip of apple roots than of annual roots. Casparian strips and lignified thickenings differentiate in the anticlinal walls of all endodermal andphi layer cells respectively, 4–5 mm from the root tip. 16 mm from the root tip and only in the endodermis opposite the phloem poles, suberin lamellae are laid down on the inner surface of the cell walls, followed 35 mm from the root tip by an additional cellulosic layer. Coincidentally with this last development, the suberin and cellulose layers detach from the outer tangential walls and the cytoplasm fragments. 85 mm from the root tip the xylem pole endodermis (50% of the endodermis) develops similarly, but does not collapse. 100–150 mm from the root tip, the surface colour of the root changes from white to brown, a phellogen develops from the pericycle and sloughing of the cortex begins. A few secondary xylem elements are visible at this stage.Plasmodesmata traverse the suberin and cellulose layers of the endodermis, but their greater frequency in the outer tangential and radial walls of thephi layer when compared with the endodermis suggests that this layer may regulate the inflow of water and nutrients to the stele.     

Fourier transform infrared-spectroscopic characterisation of isolated endodermal cell walls from plant roots: chemical nature in relation to anatomical development

The chemical nature of enzymatically isolated endodermal cell walls from Cicer arietinum L., Clivia miniata Reg. and Iris germanica L. was studied by FTIR (Fourier transform infrared) spectroscopy. Observed frequencies were assigned to functional groups present in the cell wall and relative amounts of the biopolymers suberin and lignin, cell wall carbohydrates and proteins were determined. Infrared absorption spectra indicated structural characteristics for the three different developmental states of the isolated endodermal cell wall: primary endodermis with Casparian strips (state I), secondary endodermis with suberin lamellae (state II), and tertiary endodermis with U-shaped cell wall depositions (state III). The data obtained from this study are compared with previous results obtained by chemical degradation of isolated endodermal cell walls and subsequent determination of monomeric degradation products by gas chromatography and mass spectrometry. It is concluded that FTIR spectroscopy represents a direct and nondestructive method suitable for the rapid investigation of isolated plant cell walls. Furthermore, the observation that the suberin-assigned absorption bands disappeared after transesterification of the samples with BF₃-methanol confirmed that suberin is completely degraded by this treatment. Received: 20 February 1999 / Accepted: 25 May 1999     

Structure and chemical composition of endodermal and rhizodermal/hypodermal walls of several species

CWM, isolated cell wall material
ECW, isolated endodermal cell walls
G, guaiacyl monomer
H, p-hydroxyphenyl monomer
HCW, isolated hypodermal cell walls
RHCW, isolated rhizodermal and hypodermal cell walls
S, syringyl monomer
XV, isolated xylem vessels

Endodermal cell walls of the three dicotyledoneous species Pisum sativum L., Cicer arietinum L. and Ricinus communis L. were isolated enzymatically and analysed for the occurrence of the biopolymers lignin and suberin. From P. sativum, endodermal cell walls in their primary state of development (Casparian strips) were isolated. Related to the dry weight, these isolates contained equal amounts of suberin (2·5%) and lignin (2·7%). In contrast, the endodermal cell walls of C. arietinum and R. communis, which were nearly exclusively in their secondary state of development, contained significantly higher proportions of suberin (10–20%) and only traces of lignin (1–2%). The results of the chemical analyses were supported by a microscopic investigation of Sudan III-stained root cross-sections, showing a Casparian strip restricted to the radial walls of the endodermis of P. sativum and well-pronounced red suberin lamellae in C. arietinum and R. communis roots. Compared with recently investigated monocotyledoneous species, higher amounts of suberin by one order of magnitude were detected with the secondary state of development of dicotyledoneous species. Furthermore, the carbohydrate and protein contents of primary (Clivia miniata Reg. and Monstera deliciosa Liebm.), secondary (C. arietinum and R. communis) and tertiary endodermal cell walls (Allium cepa L. and Iris germanica L.) were determined. The relative carbohydrate content of secondary endodermal cell walls was low (14–20%) compared with the content of primary (42–50%) and tertiary endodermal cell walls (60%), whereas the protein content of isolated endodermal cell walls was high in primary (13%) and secondary (8%) and low in tertiary endodermal cell walls (0·9–2%). The results presented here indicate that the quantitative chemical composition of primary, secondary, and tertiary endodermal cell walls varies significantly. Finally, cell wall proteins are described as an additional important constituent of endodermal cell walls, with the highest concentrations occurring in primary (Casparian strips) and secondary endodermal cell walls.     

Casparian strips in needles are more solute permeable than endodermal transport barriers in roots of Pinus bungeana

The structure and development of endodermal Casparian strips in Pinus bungeana needles and roots were studied by scanning electron microscopy and fluorescence microscopy. Primary pit fields (PFs) frequently occurred in radial walls of Casparian strips isolated from needles, whereas PFs were never detected in Casparian strips from roots. Formation of Casparian strips in needles as well as roots started at the outer parts of the radial walls and they finally occupied the entire radial walls of the endodermis. Fourier transform infrared (FTIR) spectroscopy of Casparian strips isolated from roots revealed significant absorption bands characteristic for suberin. However, in Casparian strips of needles, evidence for suberin was rarely detected by FTIR spectroscopy. The apoplastic permeability of Casparian strips in needles and roots was probed by the apoplastic tracers calcofluor and berberine. Casparian strips in roots efficiently blocked the apoplastic transport (AT) of calcofluor and berberine. Casparian strips in needles blocked the AT of calcofluor, but diffusion of berberine was not inhibited and berberine thiocyanate crystals were detectable in the vascular tissue of the needles. From the data presented, it must be concluded that Casparian strips in needles, which are characterized by the absence of suberin, are more solute permeable compared with Casparian strips in roots.     

Growth and Differentiation of Root Endodermis in Primula acaulis Jacq.

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

Chemical composition and ultrastructure of broad bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.) nodule endodermis in comparison to the root endodermis

Ultrastructure and development of apoplastic barriers within indeterminate root nodules formed by Vicia faba L. were examined by light and electron microscopy. The nodule outer cortex is separated from the inner cortex by a heavily suberized nodule endodermis, which matures in submeristematic regions and possesses suberin lamellae. Unsuberized passage cells are present near vascular strands, which are surrounded by a vascular endodermis attached on the inner side of the nodule endodermal cell walls. The vascular endodermis appears immediately below the meristematic apex in developmental state I (Casparian bands), gradually develops suberin lamellae, and attains developmental state II at the base of the nodule. For chemical analysis apoplastic barrier tissues were dissected after enzymatic digestion of non-impregnated tissues. Root epidermal and endodermal cell walls as well as nodule outer cortex could be isolated as pure fractions; nodule endodermal cell walls could not be separated from vascular endodermal cell walls and enclosed xylem vessels. Gas chromatography-flame ionization detection and gas chromatography-mass spectrometry were applied for quantitative and qualitative analysis of suberin and lignin in isolated cell walls of these tissues. The suberin content of isolated endodermal cell walls of nodules was approximately twice that of the root endodermal cell walls. The suberin content of the nodule outer cortex and root epidermal cell walls was less than one-tenth of that of the nodule endodermal cell wall. Substantial amounts of lignin could only be found in the nodule endodermal cell wall fraction. Organic solvent extracts of the isolated tissues revealed long-chain aliphatic acids, steroids, and triterpenoid structures of the lupeol type. Surprisingly, extract from the outer cortex consisted of 89% triterpenoids whereas extracts from all other cell wall isolates contained not more than 16% total triterpenoids. The results of ultrastructural and chemical composition are in good correspondence and underline the important role of the examined tissues as apoplastic barriers.     

Chemical analysis and immunolocalisation of lignin and suberin in endodermal and hypodermal/rhizodermal cell walls of developing maize (Zea mays L.) primary roots

The composition of suberin and lignin in endodermal cell walls (ECWs) and in rhizodermal/hypodermal cell walls (RHCWs) of developing primary maize (Zea mays L.) roots was analysed after depolymerisation of enzymatically isolated cell wall material. Absolute suberin amounts related to root length significantly increased from primary ECWs (Casparian strips) to secondary ECWs (suberin lamella). During further maturation of the endodermis, reaching the final tertiary developmental state characterised by the deposition of lignified secondary cell walls (u-shaped cell wall deposits), suberin amounts remained constant. Absolute amounts of lignin related to root length constantly increased throughout the change from primary to tertiary ECWs. The suberin of Casparian strips contained high amounts of carboxylic and 2-hydroxy acids, and differed substantially from the suberin of secondary and tertiary ECWs, which was dominated by high contents of ω-hydroxycarboxylic and 1,ω-dicarboxylic acids. Furthermore, the chain-length distribution of suberin monomers in primary ECWs ranged from C₁₆ to C₂₄, whereas in secondary and tertiary ECWs a shift towards higher chain lengths (C₁₆ to C₂₈) was observed. The lignin composition of Casparian strips (primary ECWs) showed a high syringyl content and was similar to lignin in secondary cell walls of the tertiary ECWs, whereas lignin in secondary ECWs contained higher amounts of p-hydroxyphenyl units. The suberin and lignin compositions of RHCWs rarely changed with increasing root age. However, compared to the suberin in ECWs, where C₁₆ and C₁₈ were the most prominent chain lengths, the suberin of RHCWs was dominated by the higher chain lengths (C₂₄ and C₂₆). The composition of RHCW lignin was similar to that of secondary-ECW lignin. Using lignin-specific antibodies, lignin epitopes were indeed found to be located in the Casparian strip. Surprisingly, the mature suberin layers of tertiary ECWs contained comparable amounts of lignin-like epitopes. Received: 19 August 1998 / Accepted: 3 February 1999     

Casparian strips in needles of Pinus bungeana: isolation and chemical characterization

By using cell wall degrading enzymes, Casparian strips were for the first time isolated from Pinus bungeana needle endodermis. They appeared as a fine network, similar to those isolated from roots. Fourier transform infrared spectroscopic analysis provided evidence that the Casparian strips were impregnated with lignin, suberin, cellulose and cell wall proteins.     

Comparative investigation of primary and tertiary endodermal cell walls isolated from the roots of five monocotyledoneous species: chemical composition in relation to fine structure

The chemical composition of isolated endodermal cell walls from the roots of the five monocotyledoneous species Monstera deliciosa Liebm., Iris germanica L., Allium cepa L., Aspidistra elatior Bl. and Agapanthus africanus (L.) Hoffmgg. was determined. Endodermal cell walls isolated from aerial roots of M. deliciosa were in their primary developmental state (Casparian bands). They contained large amounts of lignin (6.5% w/w) and only traces of suberin (0.5% w/w). Endodermal cell walls isolated from the other four species were in their tertiary developmental state. Lignin was still the more abundant cell wall polymer with amounts ranging from 3.8% (w/w, A. cepa) to 4.5% (w/w, I. germanica). However, compared to endodermal cell walls in their primary state of development (Casparian bands), tertiary endodermal cell walls contained significantly higher amounts of suberin, ranging from 1.8% (w/w, I. germanica) to 3.0% (w/w, A. africanus). Thus, chemical characterization of endodermal cell walls from five different species revealed that lignin was the dominant cell wall polymer in the Casparian band of M. deliciosa, whereas tertiary endodermal cell walls contained, in addition to lignin, increasing amounts of suberin (I. germanica, A. cepa, A. elatior and A. africanus). Besides the two biopolymers lignin and suberin, cell wall carbohydrates in the range of between 40 and 60% were also quantified. The sum of all cell wall compounds investigated by gas chromatography resulted in a recovery of 50–80% of the dry weight of the isolated cell wall material. Quantitative chromatographic results in combination with microscopic studies are consistent with the existence of a distinct suberin lamella and lignified tertiary wall deposits. From these data it can be concluded that the barrier properties of the endodermis towards the apoplastic transport of ions and water will increase from primary to tertiary endodermal cell walls due to their increasing amounts of suberin. Received: 23 August 1997 / Accepted: 28 January 1998     

Root Endodermis and Exodermis: Structure, Function, and Responses to the Environment

Roots of virtually all vascular plants have an endodermis with a Casparian band, and the majority of angiosperm roots tested also have an exodermis with a Casparian band. Both the endodermis and exodermis may develop suberin lamellae and thick, tertiary walls. Each of these wall modifications has its own function(s). The endodermal Casparian band prevents the unimpeded movement of apoplastic substances into the stele and also prevents the backflow of ions that have moved into the stele symplastically and then were released into its apoplast. In roots with a mature exodermis, the barrier to apoplastic inflow of ions occurs near the root surface, but prevention of backflow of ions from the stele remains a function of the endodermis. The suberin lamellae protect against pathogen invasion and possibly root drying during times of stress. Tertiary walls of the endodermis and exodermis are believed to function in mechanical support of the root, but this idea remains to be tested. During stress, root growth rates decline, and the endodermis and exodermis develop closer to the root tip. In two cases, stress is known to induce the formation of an exodermis, and in several other cases to accelerate the development of both the exodermis and endodermis. The responses of the endodermis and exodermis to drought, exposure to moist air, flooding, salinity, ion deficiency, acidity, and mechanical impedance are discussed.     

Root Endodermis and Exodermis: Structure,Function, and Responses to the Environment

Roots of virtually all vascular plants have an endodermis with a Casparian band, and the majority of angiosperm roots tested also have an exodermis with a Casparian band. Both the endodermis and exodermis may develop suberin lamellae and thick, tertiary walls. Each of these wall modifications has its own function(s). The endodermal Casparian band prevents the unimpeded movement of apoplastic substances into the stele and also prevents the backflow of ions that have moved into the stele symplastically and then were released into its apoplast. In roots with a mature exodermis, the barrier to apoplastic inflow of ions occurs near the root surface, but prevention of backflow of ions from the stele remains a function of the endodermis. The suberin lamellae protect against pathogen invasion and possibly root drying during times of stress. Tertiary walls of the endodermis and exodermis are believed to function in mechanical support of the root, but this idea remains to be tested. During stress, root growth rates decline, and the endodermis and exodermis develop closer to the root tip. In two cases, stress is known to induce the formation of an exodermis, and in several other cases to accelerate the development of both the exodermis and endodermis. The responses of the endodermis and exodermis to drought, exposure to moist air, flooding, salinity, ion deficiency, acidity, and mechanical impedance are discussed.     

植物内皮层的分化及其屏障功能研究进展

植物根系最主要的作用之一是从土壤中获取养分并将其运输至地上部。水和营养物质径向穿过根的表皮、皮层、内皮层等所有外部细胞层,才能到达中柱,以供地上部代谢所需。其中,内皮层细胞在发育过程中会经历两个特殊的分化阶段,分别形成凯氏带和木栓层两种扩散屏障,二者在控制养分获取与流失方面起着重要的作用。该文就近年来国内外有关植物内皮层分化过程及其屏障功能方面的研究进展进行了综述,以期对深入探索内皮层屏障在植物生长发育和逆境适应中的作用提供参考,为植物育种工作开辟新的思路。     

Radial widening of the Casparian strip follows induced radial expansion of endodermal cells

The Casparian strip, the barrier to apoplastic transport that is located at the endodermis in roots and stems, is formed by individual endodermal cells and is constructed as a highly organized mesh within the primary wall. Since little is known about the mechanism of formation of the strip, we tried to obtain morphological evidence for the existence, prior to suberization and lignification, of some regulatory system at the expected site of the strip. Endodermal cells in etiolated pea stems were induced to expand in the radial direction by piercing the stems through the cortex before formation of the strip. The radial width of the strip increased significantly with the expansion of the radial walls of these endodermal cells. The expansion of the cells occurred before the formation of the strip. However, strips that had already been formed when the stems were pierced did not increase in width despite an induced expansion of the radial walls. These observations suggest that some positional information exists in the radial wall of endodermal cells that defines the future site of formation of the strip and its width.     

OBSERVATIONS ON THE ROOT ANATOMY OF RICE (ORYZA SATIVA L.)

Rice plants were grown hydroponically and roots were prepared for light and electron microscopy using standard techniques. The roots are bounded by an epidermis, exodermis, and fibrous layer. The exodermis has a suberin lamella along its inner tangential wall. The fibrous layer is composed of thick-walled lignified cells with little pitting. The cortical parenchyma is compact when young, but expands and separates to form a zone of cell walls and air spaces in a spoked arrangement. Supporting columns of living parenchyma cells are occasionally present, particularly near lateral roots. The endodermis is typical for grasses with Casparian strips, suberin lamellae, and tertiary state walls with numerous pits. The pericycle and pith become sclerified. Protoxylem elements alternate with protophloem in the young root; later, early metaxylem, late metaxylem, and metaphloem proliferate. The exodermis, fibrous layer, lacunate cortex, and endodermis appear to present a formidable barrier to radial ion movement in the mature portions of the root.     

Casparian strip development and its potential function in salt tolerance

The root system is particularly affected by unfavourable conditions because it is in direct contact with the soil environment. Casparian strips, a specialised structure deposited in anticlinal walls, are characterised by the impregnation of the primary wall pores with lignin and suberin. The Casparian strips in the endo- and exodermis of vascular plant roots appear to play an important role in preventing the non-selective apoplastic bypass of salts into the stele along the apoplast under salt stress. However, only a few investigations have examined the deposition and function of these apoplastic barriers in response to salt stress in higher plants.     

Ultrastructure and functioning of the transport system of the leguminous root nodule

Summary The structure of the vascular tissues of nitrogen-fixing nodules of 27 genera of legumes and some non-legumes has been investigated by light microscopy. Pisum and Trifolium nodules have been examined by electron microscopy.Attention is directed to the presence of a pericycle in the vascular bundles of the nodules. In 7 of the legumes the pericycle cells possess a wall labyrinth consisting of branched filiform protuberances. The ultrastructure of the pericycle cell cytoplasm is described: its most striking feature is its abundant rough endoplasmic reticulum. These cells surround the xylem and phloem of the bundles, and are in turn surrounded by a layer of endodermal cells with Casparian strips. The pericycle cells develop their wall labyrinth in the levels of the nodule at which the bacterial tissue becomes pigmented; in nodule senescence their cytoplasm is disrupted level with the breakdown of the bacterial tissue.A pathway for symplastic lateral transfer of assimilates exists, from the sieve elements through the pericycle, endodermis and cortex to the bacterial tissue. The apoplast within the endodermis consists largely of the pericycle wall labyrinth and the xylem. The ultrastructure of the Casparian strip resembles that of roots.Intact, detached nodules can be induced to bleed a fluid from their severed vascular tissue. This fluid is exceptionally rich in organic nitrogen, particularly amides, but does not appear to contain sugars. Comparison between its amino acid composition and that of other parts of the nodule suggests that an active uptake or secretion of nitrogenous compounds precedes export from the nodule. Special functions are suggested for the nodule endodermis and the pericycle cells in this export process.     

Water Pathways in Higher Plants: II. WATER PATHWAYS IN BOOTS

The technique for studying the pathways of water movement described,in the first paper of this series has been applied to the transpirationstream in the roots of higher plants. Free space is confinedto the cell walls except in flooded tissue where intercellularspaces are also included. Errors involved in free-space estimatesare discussed and free-space volumes of 5.4 per cent have beenobtained for wheat root cortex and 4.1 per cent for carrot xylemparenchyma. The main water-absorbing regions of roots beginimmediately behind the elongating zone, where the first xylemelements are fully differentiated, and end when the endodermisundergoes secondary wall development. In the cortex the transpirationstream is located mainly in the cell wall. Calculations indicatethat the symplastic pathway is of only minor importance in transpiringplants. At the endodermis the free-space pathway is blockedby the Casparian strip and all water and solute entering thestele must pass through the lumen of the endodermal cells. Thepermeability of individual endodermal cells varies considerablyboth between cells of the same species and between those ofdifferent species. Once inside the endodermis, the transpirationstream returns to the cell-wall pathway until it reaches thexylem vessels where it enters the lumen of the mature xylemelements.