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Keywords:

  • Cicer arietinum L.;
  • Fabaceae;
  • Pisum sativum L.;
  • Fabaceae;
  • Ricinus communis L.;
  • Euphorbiaceae;
  • apoplast;
  • Casparian strip;
  • cell wall carbohydrates;
  • cell wall proteins;
  • endodermis;
  • lignin;
  • root;
  • suberin;
  • suberin lamella

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. References

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.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. References

The nutrient and water supply of higher, land-living plants is normally accomplished by the roots [see Marschner (1995)]. Substances have to move from the soil solution radially through the root cortex into the central cylinder where xylem elements, which perform the long-distance transport to the shoots, are located. In order to subject nutrient uptake to a cytoplasmic control, the apoplastic space of the root, which otherwise would form a continuous aqueous connection between the soil and the xylem vessels, must be blocked. This important function of an apoplastic transport barrier is carried out by the endodermis, which forms a layer of intercellular space-free cells separating the cortex from the central cylinder [see Esau (1977)].

The function of the endodermis as an apoplastic transport barrier is based on a distinct structural development of the endodermal cell walls (Kroemer 1903). In its primary state of development (Casparian strips), radial endodermal cell walls are impregnated with substances described as suberin and/or lignin (van Fleet 1961; von Guttenberg 1968; Karahara & Shibaoka 1994). In its secondary state of development, a suberin lamella is deposited on the primary cell wall, coating all endodermal cell walls. Finally, in its third state of development, a thick deposition of lignified cellulose walls is added, often only to the radial and inner tangential walls resulting in a U-shaped appearance of the endodermal cells.

Endodermal cell wall anatomy and development has been analysed and described in detail by light microscopy and histochemistry since its first description by Caspary in 1856 up to now (Kroemer 1903; Wilson & Peterson 1983). The chemical composition of the endodermal cell wall in etiolated stems of Sorghum seedlings was described to contain suberin (Espelie & Kolattukudy 1979). In order to improve our knowledge of the chemical composition of the endodermal cell wall of roots, which in turn will contribute to an improved understanding of its barrier properties, we started to investigate endodermal cell walls by modern chemical analytical methods. Endodermal cell walls (ECW) were isolated enzymatically as described recently (Robards, Payne & Gunning 1976; Karahara & Shibaoka 1992; Schreiber et al. 1994) and chemically degraded by various methods. Gas chromatographic analysis of the degradation products allowed direct determination of the suberin, lignin, and carbohydrate monomers occurring in ECW of various plant species (Schreiber 1996; Zeier & Schreiber 1997, 1998).

In the past, only root samples from only monocotyledoneous plant species were investigated. Because these do not have secondary growth, the endodermis is retained and the amounts of ECW necessary for chemical analyses are easily isolated. For the present study, we succeeded in isolating sufficient amounts of ECW from dicotyledoneous plants. This allowed the investigation of the chemical composition of ECW to be extended to include three dicotyledoneous species and to compare them with monocotyledoneous species.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. References

Root samples

Seeds of Pisum sativum L. (cv. Alaska) were germinated and cultivated in moist vermiculite for 8 d before the roots were sampled. Cicer arietinum L. (unknown cultivar) and Ricinus communis L. (cv. Sanguines) were germinated in moist vermiculite for 4 and 12 d, respectively. Cultivation of these two species was continued on hydroponic solution for 4 weeks before taking the root samples. The concentrations of macronutrients (1500 mmol m–3 Ca(NO3)2, 1000 mmol m–3 KCl, 1250 mmol m–3 KNO3, 750 mmol m–3 MgSO4, 375 mmol m–3 KH2PO4, 126 mmol m–3 NaFeEDTA) and micronutrients (68·9 mmol m–3 H3BO4, 13·7 mmol m–3 MnCl2, 1·5 mmol m–3 ZnCl2, 1 mmol m–3 Na2MoO4, 0·5 mmol m–3 CuCl2) in the hydroponic solution were one quarter the concentrations recommended by Hoagland & Arnon (1938). Adventitious roots from Allium cepa L. were obtained by cultivating onion bulbs for 6 weeks above water-filled Erlenmeyer flasks. Roots from full-grown plants of Clivia miniata Reg. and Iris germanica L., and aerial roots from a full-grown Monstera deliciosa Liebm. were obtained from the Botanical Garden in Würzburg.

Isolation of cell wall material (CWM) from roots

The enzymatic isolation of the different cell walls has been previously described in detail (Karahara & Shibaoka 1992; Schreiber et al. 1994). Root segments (50 mm) were incubated in an enzymatic solution of cellulase (Onozuka R-10, Serva, Heidelberg, Germany) and pectinase (Macerozyme R-10, Serva). After several days, cell walls from different root tissues which had resisted the enzymatic attack could be separated. Xylem vessels (XV) and ECW were separated from central cylinders. The outermost layer of the roots was a rhizodermis. Because both rhizodermal and attached hypodermal cell walls resisted the enzymatic attack and did not separate from each other, a cell wall fraction called RHCW (isolated rhizodermal and hypodermal cell walls) was isolated. Isolated CWM was thoroughly washed using borate buffer (10 mol m–3, pH 9) and deionized water, dried and stored over silica gel until further use.

Microscopy

Light and fluorescence microscopic investigations of root samples from different plant species have been described in detail by Zeier & Schreiber (1998). Root sections (5 mm) were taken at different distances from the root tip and fixed for 24 h in a phosphate-buffered saline (PBS)-buffered solution (pH 7·2) of formaldehyde (37 kg formaldehyde m–3). Sections of 15–25 μm were cut at – 25 °C using a cryomicrotome (Cryostat H 500 M, Microm), transferred to glass slides, and mounted in glycerol/water (1 volume glycerol:1 volume water). The samples were examined using an Axioplan microscope (Zeiss, Oberkochen, Germany) with conventional bright field illumination or fluorescence excitation at 365 nm (Zeiss filter sets number 01). Staining with Sudan III was carried out according to Gerlach (1984).

Chemical degradation of isolated cell walls

After a thorough extraction with chloroform/water (1 volume chloroform:1 volume water), isolated CWM was subjected to various chemical degradation methods specific for the detection of lignin, suberin, cell wall carbohydrates and cell wall proteins. Details of the lignin, suberin and carbohydrate analyses have been described by Zeier & Schreiber (1998). Thioacidolysis was used for the detection of lignin according to Lapierre, Pollet & Monties (1991). Suberin was analysed after the transesterification of isolated CWM according to Kolattukudy & Agrawal (1974). Cell wall carbohydrates were analysed according to Blakeney et al. (1983). In order to characterize the cell wall protein content, ECW (2 mg) was hydrolysed under an atmosphere of argon (6 kmol HCl m–3, 130 °C, 24 h). After cooling, the reaction mixture was filtered and the hydrochloric acid was evaporated under vacuum. In order to remove remaining traces of HCl completely, the residue was redissolved in water (1 cm3) and dried again. This procedure was repeated three times before dissolving the residue in 160 mm3 of sample buffer (Li3citrate 100 mol m–3; citric acid 68·5 mol m–3; 20 cm3 Bis-(2-hydroxyethylsulphide) adjusted to pH 2·2 using HCl.

Chromatographic analyses of the degradation products

Gas chromatographic analyses and mass spectrometric identification of the chloroform/methanol extracts and of lignin, suberin and carbohydrate monomers were performed as previously described in detail (Zeier & Schreiber 1997). Quantitative sample analyses were carried out on a gas chromatograph (HP 5890 Series II gas chromatograph, Hewlett-Packard, CA, USA) equipped with a flame ionization detector. Qualitative sample analyses were performed by gas chromatography (HP 5890 Series II gas chromatograph, Hewlett-Packard) combined with a quadrupole mass selective detector (HP 5971A mass selective detector, Hewlett-Packard). A 150 mm3 fraction of the sample buffer containing the amino acids of the hydrolysed cell wall proteins was analysed by high-performance liquid chromatography (HPLC; LC 5001 Biotronic, Eppendorf, Hamburg, Germany). Amino acids, which were identified according to their retention times, were detected as ninhydrin derivatives at 570 nm with the exception of proline and OH-proline, which were detected at 440 nm.

Reproducibility

The cell wall samples used in this investigation represent the mean of a large number of individual plants as they were isolated from at least 15 individual plants. The analysis of the cell wall isolates with each of the different methods was performed twice. The results are given as means of the two independent repetitions. Data given as percentage values are always based on dry matter. Reproducibility was good and deviations between the two repetitions scarcely exceeded 10% and were never higher than 20%.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. References

Microscopic investigation of the endodermis

Light microscopic investigation of P. sativum roots, which were used for the isolation of ECW, revealed that the endodermis was essentially in its primary state of development. Casparian strips, which were detectable as small dots in the radial walls of the endodermal cells, were slightly stained by Sudan III (Fig. 1a). Fluorescence microscopy at an excitation wavelength of 365 nm revealed a well-pronounced autofluorescence of the Casparian strips in the blue (Fig. 1b). The roots of C. arietinum and R. communis, which were sampled for the isolation of ECW, were nearly exclusively in their secondary state of development, as revealed by the light microscopic investigation of Sudan III-stained cross-sections (Fig. 1c & e). A well-pronounced red-coloured suberin layer deposited in the inner radial and tangential cell walls of the endodermal cells was discernible over nearly the complete root length (Fig. 1c & e). Excitation of C. arietinum root cross-sections at 365 nm showed an intensive blue autofluorescence of the ECW and XV located in the central cylinder of the root (Fig. 1d). Microscopic investigations of hypodermal and rhizodermal layers of the three species showed only a weak autofluorescence and a suberin lamella after staining with Sudan III was only visible in the hypodermis of R. communis (data not shown). Casparian bands were not detected in the hypodermis of the three species.

image

Figure 1. . Bright field and fluorescence microscopic pictures of root cross-sections of the three species Pisum sativum, Cicer arietinum and Ricinus communis. (a) Bright field microscopic picture of a root cross-section of P. sativum indicating the primary developmental state of the endodermis. Casparian strips, stained with Sudan III, are only discernible as tiny red dots in the radial walls of the endodermal cells (black arrows). (b) Autofluorescence of Casparian strips in a cross-section of P. sativum root after excitation at 365 nm. (c) Cross-section of C. arietinum root indicating the secondary state of endodermal development. Sudan III-stained suberin is deposited as a continuous lamella on the inner sides of the radial and tangential walls of the endodermal cells. (d) Blue autofluorescence of Casparian bands and suberin lamellae in endodermal cell walls, and xylem vessels in a cross-section of C. arietinum root after excitation at 365 nm. (e) Bright field microscopic picture of a cross-section of R. communis root indicating the secondary state of endodermal development. The suberin lamellae, which are stained with Sudan III, are deposited on the inner walls of the endodermal cells.

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Chemical characterization of suberin

Related to a dry matter basis, ester-linked aliphatic suberin components amounted to 2·5% in Casparian strips of P. sativum (Fig. 2). ECW of C. arietinum and R. communis in their secondary state of development released 19·5% and 9·8% of aliphatic suberin monomers, respectively. Ester-linked aliphatics of suberin origin were also detected in RHCW, ranging from 0·5% to 4·7% (Fig. 2). Suberin contents of RHCW were always significantly lower compared with ECW (Fig. 2). XV released only traces of aliphatic suberin monomers (P. sativum and R. communis; Fig. 2) or no suberin monomers at all (C. arietinum; Fig. 2).

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Figure 2. . Total amounts of suberin and lignin from rhizodermal and hypodermal cell walls (RHCW), endodermal cell walls (ECW) and xylem vessels (XV) of the three investigated species Pisum sativum, Cicer arietinum and Ricinus communis obtained after BF3/MeOH-transesterification and thioacidolysis, respectively. The amounts are given as μg suberin or lignin per mg dry weight of isolated wall samples.

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A closer look at the nature of the released suberin monomers revealed that ω-hydroxycarboxylic acids (45–60%) followed by 1,ω-dicarboxylic acids (25–35%) were the most dominant substance classes in ECW of all species investigated (Fig. 3a). Carboxylic acids (10–15%), 2-hydroxycarboxylic acids (0–9%) and alcohols (0–3%) occurred in lower amounts (Fig. 3a). A suberin composition different from ECW was found in RHCW of P. sativum and C. arietinum (Fig. 3b). Carboxylic acids (55–70%) and 2-hydroxyacids (25–30%) were the dominant compounds, whereas ω-hydroxyacids and 1,ω-diacids were not present (P. sativum) or only minor components (C. arietinum) in RHCW (Fig. 3b). RHCW isolated from R. communis showed a suberin composition comparable to ECW (Fig. 3a & b). The chain length distribution of the long-chain aliphatic suberin components in ECW ranged from C16 to C24 for P. sativum (Fig. 4a). Palmitic (C16) and oleic (C18(1)) acid derivatives were the dominant compounds. Further maxima occurred at C20 for P. sativum and C. arietinum (Fig. 4a & b) and at C22 for R. communis (Fig. 4c).

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Figure 3. . Substance classes of aliphatic suberin components detected in isolated endodermal cell walls (a) and in rhizodermal and hypodermal cell walls (b) of the three investigated species Pisum sativum, Cicer arietinum and Ricinus communis. The percentage values were calculated from the total amount of aliphatic suberin.

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Figure 4. . Chain length distributions of aliphatic suberin components released by BF3/MeOH-transesterification from endodermal cell walls of the three investigated species Pisum sativum (a), Cicer arietinum (b) and Ricinus communis (c). The amounts are given as μg suberin per mg dry weight of isolated cell wall samples.

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Significant amounts (3%) of non-esterified carboxylic acids were detected in ECW of C. arietinum (Fig. 5). The chain length distribution of the free acids ranging from C16 to C25 (Fig. 5) was comparable to the distribution of the esterified suberin components (Fig. 4b). In addition to free carboxylic acids, the extract of ECW of C. arietinum released detectable amounts of the phytosterols campesterol, stigmasterol and β-sitosterol (data not shown).

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Figure 5. . Chain length distribution of long-chain, carboxylic acids released after extraction of isolated endodermal cell walls of Cicer arietinum with chloroform/methanol. The amounts are given as μg acid per mg dry weight of isolated cell wall samples.

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Chemical characterization of lignin

The dominating lignin degradation products were the trithioethylated aromatic monomers corresponding to the three lignin units p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S). The lignin content was determined by combining the three H, G, S monomers (Fig. 2). The lignin content was highest in Casparian strips of P. sativum (2·7%) and decreased in secondary ECW from 2·1% in C. arietinum to 0·6% in R. communis (Fig. 2). Compared with ECW, the lignin content was lower in RHCW of all investigated species, ranging from 0·4% to 1·1% (Fig. 2). The highest amounts of lignin-derived degradation products (6–10%) were released from XV (Fig. 2).

The H/G/S ratios of the detected lignin monomers revealed that G-units represented the most dominant constituents in all species and in all different cell wall samples (Table 1). The lignin of XV contained only traces of H-units (1%) and, depending on the species, varying amounts of S-units (3–42%) were detected (Table 1). In endodermal lignin, S-units ranged from 5% to 25% and a low H content (2–9%) was observed (Table 1). RHCW of P. sativum showed the highest amounts of H (13%), whereas in RHCW of C. arietinum and R. communis only traces of H-units were detected (Table 1). Apart from the dominant G-units in RHCW, S-units varied from 16% to 44% (Table 1).

Table 1.  . Hydroxyphenyl (H), guaiacyl (G) and syringyl (S) ratios of lignins from rhizodermal and hypodermal cell walls (RHCW), endodermal cell walls (ECW) and xylem vessels (XV) isolated from the three different species Pisum sativum, Cicer arietinum and Ricinus communisThumbnail image of

Chemical characterization of cell wall carbohydrates

Acid hydrolysis of polysaccharides yielded alditol acetates originating from the common cell wall monosaccharides rhamnose, arabinose, xylose, mannose, glucose and galactose (Table 2). The carbohydrate content was 42–50% in primary ECW of C. miniata and M. deliciosa, 14–20% in secondary ECW of C. arietinum and R. communis and 62% in tertiary ECW of A. cepa and I. germanica (Table 2). Arabinose and glucose represented the predominant monosaccharides in primary and secondary ECW, whereas in tertiary ECW glucose and xylose were the main constituents (Table 2).

Table 2.  . Monosaccharides released by acid hydrolysis from primary, secondary and tertiary endodermal cell walls isolated from the six species Clivia miniata, Monstera deliciosa, Cicer arietinum, Ricinus communis, Allium cepa and Iris germanica. The results are given in μg per mg dry weight of isolated cell wall samples Thumbnail image of

Chemical characterization of cell wall proteins

The protein content of the samples was estimated by combining all obtained amino acids released after hydrolysis with 6 N HCl (Table 3). The highest amounts of wall-associated protein were found in primary (13–14%) and secondary ECW (8–20%), whereas protein contents in tertiary ECW amounted to only 1–2% (Table 3). Significant amounts of hydroxyproline (4–17 Mol%), serine (6–11 Mol%), lysine (5–8 Mol%), proline (3–8 Mol%), glycine (9–25 Mol%) and valine (5–8 Mol%) were detected in isolated CWM of all investigated species (Table 3).

Table 3.  . Amino acids released by acid hydrolysis from primary, secondary and tertiary endodermal cell walls isolated from the six species Clivia miniata, Monstera deliciosa, Cicer arietinum, Ricinus communis, Allium cepa and Iris germanica. Individual amino acids are given in Mol%, whereas the total of the amino acids is given in μg per mg dry weight of isolated cell wall samples Thumbnail image of

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. References

Developmental states of the investigated ECW from dicotyledoneous species

As revealed by light microscopy, the ECW of P. sativum was in its primary developmental state (Fig. 1a & b), whereas ECW from the other two species, C. arietinum and R. communis, were nearly exclusively in their secondary developmental state (Fig. 1c–e). Because ECW was isolated from very young P. sativum plants, the endodermis was in its primary developmental state. A transition to the secondary state of development occurred near the root base and this zone was not used for the isolation of ECW.

With the other two species, roots were sampled from significantly older plants having a root length of 40–50 cm. Here the primary developmental state was present within the first 10 cm of the roots, and the remaining 40 cm of the roots was in the secondary developmental state. Because complete roots were used for the isolation of ECW, the material obtained was in fact a mixture of the primary and the secondary developmental states. However, it is reasonable to argue that the results obtained with the two species C. arietinum and R. communis essentially reflected the chemical composition of the secondary developmental state, as more than 80% of the root material was characterized by a suberin lamella. Thus, the ECW isolated from the three different dicotyledoneous plant species allowed the comparison of the Casparian strip with the secondary developmental state of the endodermis.

Comparison of suberin and lignin in ECW

Until now, Casparian strips (primary developmental state) of the two monocotyledoneous species C. miniata and M. deliciosa had revealed that lignin was the dominant biopolymer (5–6%), whereas only traces (0·1–1%) of suberin were detected (Zeier & Schreiber 1997, 1998). Suberin amounts in ECW of P. sativum were low (Fig. 2) and in a comparable range as the amounts found in monocotyledoneous species. However, the lignin content of the Casparian strip of P. sativum was in the same range as the suberin content (Fig. 2). Therefore, lignification of the Casparian strips of the monocotyledoneous species was significantly higher compared with the dicotyledoneous species. This might be related to the further development of the endodermis of investigated species. The endodermis of the monocotyledoneous species C. miniata, as well as M. deliciosa, remains essentially in its primary developmental state and, therefore, a thorough lignification might increase its resistance towards mechanical stress and microbial attack. With the endodermis of P. sativum, changing from the primary to the secondary developmental state, as it is representative for many dicotyledoneous species (Kroemer 1903; von Guttenberg 1968; Esau 1977), this strong lignification might not be necessary.

ECW of C. arietinum and R. communis, which to our knowledge represented the first secondary ECW whose chemical composition was analysed, contained the highest amounts of suberin (10–20%; Fig. 2) compared with all other species in their primary and tertiary developmental state investigated up to now (Zeier & Schreiber 1998). This is good evidence that the Sudan III-stained lamella, which can be observed by light microscopy (Fig. 1c & e) and which was described regularly in the literature (Wilson & Peterson 1983; Barnabas & Peterson 1992), contains aliphatic suberin as a major compound. This conclusion is also supported by the fact that extremely low amounts of lignin were detected in ECW of C. arietinum and R. communis (Fig. 2). Even tertiary ECW of the four species I. germanica, A. cepa, Aspidistra elatior Bl. and Agapanthus africanus (L.) Hoffmgg., contained only between 1·5% and 3% suberin (Zeier & Schreiber 1998) compared with the two dicotyledoneous species investigated here (Fig. 2).

Looking more closely at the relative contribution of the different substance classes (Fig. 3) and the chain length distributions (Fig. 4) of the suberin, it becomes clear that they are not very different in the ECW of the three investigated species. The results obtained here are also in good agreement with the suberin compositions of six monocotyledoneous species determined recently (Zeier & Schreiber 1998). In nearly all ECW samples, derivatives of C16 and C18 aliphatics represented the most dominant suberin monomers, and chain length distributions normally ranged from C16 to C26 (Fig. 4). Thus, there is qualitatively no significant difference between the suberin composition of the ECW of mono- and dicotyledoneous species.

Comparison of suberin and lignin in RHCW, ECW and XV

As expected, XV representing lignified secondary cell walls contained significantly higher amounts of lignin (6–10%) compared with ECW and RHCW (Fig. 2). In accordance with recent investigations (Schreiber 1996; Zeier & Schreiber 1997), the G/S ratios found here were again highest in XV and they showed significantly lower values in ECW and RHCW (Table 1), indicating chemically different lignin polymers in the different cell wall fractions XV, ECW and RHCW.

Looking more closely at the suberin composition of the different cell wall fractions, it becomes evident that XV, as expected for a lignified secondary cell wall, contained no or only traces of suberin monomers (Fig. 2). Furthermore, the lignin and suberin content of RHCW was also significantly lower compared with ECW (Fig. 2). In addition, with P. sativum and C. arietinum, where extremely low suberin contents in RHCW were measured, the relative contribution of the substance classes was also very different between ECW and RHCW (Fig. 3). In RHCW, 2-hydroxacids appeared as a new dominant substance class, which was absent in ECW (Fig. 3).

The high suberin content in the ECW, together with the partially different chemical composition of RHCW compared with ECW, might indicate different functions of the two investigated cell wall fractions. Due to the high suberin content the endodermal cell wall should establish a more effective apoplastic transport barrier, whereas the function of the weakly suberized rhizodermis and hypodermis might be the formation of a barrier resistant to microbial attack. This hypothesis of the ECW forming an effective transport barrier for the passive diffusion of water and ions is further supported by the significant amounts of free carboxylic acids (3%) deposited into the ECW polymer (Fig. 5). This is supported by recent results, which demonstrated that waxes associated with suberized layers form the major diffusion barrier to water (Soliday, Kolattukudy & Davis 1979). In suberized interfaces separating plant storage organs from the soil environment, waxy compounds of different substance classes (alkanes, esters, primary alcohols and carboxylic acids) have also been detected (Espelie, Sadek & Kolattukudy 1980). The free carboxylic acids (Fig. 5), showing a comparable chain length distribution as the esterified suberin components (Fig. 4b), could also serve as monomeric precursors which are later incorporated in the suberin polymer.

Comparison of cell wall carbohydrate and protein contents of primary, secondary and tertiary ECW isolated from mono- and dicotyledoneous species

Yields of suberin and lignin analyses of ECW carried out in the past in our laboratory (Zeier & Schreiber 1997, 1998) never exceeded 10%, indicating that a substantial part of ECW must be composed of additional compounds. From the literature it is well known that suberized (Kolattukudy 1981; Stark et al. 1994; Bernards & Lewis 1998) and lignified cell walls (Lin & Dence 1992) are always associated with carbohydrates. CHN analysis of ECW from C. miniata indicated that a substantial amount of the isolated CWM might be composed of cell wall proteins, as a significant amount of N was detected (Schreiber et al. 1994). Thus, a comparative investigation of endodermal development concerning the amounts of cell wall carbohydrates (Table 2) and proteins (Table 3) in primary, secondary and tertiary ECW was performed. As typical examples for primary, ECW C. miniata (Schreiber 1996; Zeier & Schreiber 1997) and M. deliciosa (Zeier & Schreiber 1998), for secondary ECW C. arietinum and R. communis and for tertiary ECW A. cepa and I. germanica (Zeier & Schreiber 1998) were examined.

Carbohydrate analysis yielded substantial amounts of monomeric sugars ranging between 14% and 60% (Table 2). This indicates that a large fraction of the isolated ECW consists of a regular plant cell wall. Obviously, in the presence of lignin and suberin, carbohydrates are at least partially shielded from the cell wall degrading enzymes used for the isolation of ECW. The relative amounts of carbohydrates were significantly higher in primary and tertiary endodermal ECW compared with secondary ECW (Table 2). This is best explained by the way our results are expressed. Because the results were always related to the dry weight of the isolated ECW used for the analyses, a high amount of suberin, as detected with C. arietinum and R. communis (Fig. 2), must necessarily lead to a decrease of the relative amounts of the other cell wall polymers. Additionally, this allows the conclusion that the secondary ECW is dominated by the deposition of suberin over minor amounts of other cell wall polymers such as lignin or carbohydrates. The opposite is true for the tertiary ECW, where the carbohydrate content of ECW was the highest of all the three developmental states (Table 2), indicating that the characteristic U-shaped cell wall depositions were essentially composed of partially lignified plant cell walls (Table 2) without further suberin depositions, as suggested by Kroemer (1903). Furthermore, yields of amino acid analyses indicated that there must be a substantial amount of structural cell wall proteins in primary and secondary ECW but not in tertiary ECW (Table 3). The most prominent amino acids were OH-proline (4–17%) and glycine (9–25%), which are important constituents of extensins and glycine-rich proteins forming common plant cell wall proteins (Showalter 1993). Extensins are often glycolsylated with arabinose, occurring in highest concentrations in Casparian strips and constantly decreasing over secondary to tertiary ECW (Table 2). Obviously structural cell wall proteins form important constituents of Casparian strips and of secondary ECW. Due to the increase in lignified cellulose walls in the tertiary ECW, the relative content of cell wall proteins in these ECW is low (Table 3).

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. References

On the basis of the results obtained for mono- and dicotyledoneous species, a preliminary model of the primary, secondary and tertiary endodermal cell wall composition and function can be deduced. Endodermal cell walls with Casparian bands are characterized by relatively low amounts of suberin and lignin and high carbohydrate and cell wall protein contents. Secondary endodermal cell walls are characterized by a lamella of the aliphatic, lipophilic biopolymer suberin and, thus, compared with the primary endodermis they should represent a more effective barrier towards the radial diffusion of water and dissolved ions. Finally, the tertiary developmental state, which was only observed with monocotyledoneous species lacking secondary growth in the present study, is characterized by the deposition of additional, thick lignified cell walls, further increasing the mechanical stability of the endodermal cell walls, as suggested by Priestley & North (1922).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. References

Financial support by the DFG (SCHR 506/1–2, SCHR 506/5–1 and SFB 251) is gratefully acknowledged.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. References
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