Planta (2004) 219: 41–47 DOI 10.1007/s00425-004-1208-7

ORIGINAL ARTICLE

Ichirou Karahara Æ Atsuo Ikeda Æ Takanori Kondo Yuzo Uetake Development of the Casparian strip in primary of maize under salt stress

Received: 17 June 2003 / Accepted: 17 December 2003 / Published online: 19 February 2004 Ó Springer-Verlag 2004

Abstract The Casparian strip in the endodermis of vas- Keywords Casparian strip (band) Æ Salinity (salt cular plant roots appears to play an important role in stress) Æ Endodermis Æ Æ Zea preventing the influx of salts into the through the under salt stress. The effects of salinity on the development and morphology of the Casparian strip in primary roots of maize (Zea mays L.) were studied. Introduction Compared to the controls, the strip matured closer to the tip with increase in the ambient concentration Roots are supposed to take up water and necessary of NaCl. During growth in 200 mM NaCl, the number solutes from the soil, simultaneously avoiding the influx and the length of the endodermal cells in the region by diffusion of unnecessary solutes into the stele. Since between the root tip and the lowest position of the roots manage to fulfill these contradictory requirements, endodermal strip decreased, as did the apparent rate of it seems very probable that the internal tissues that are production of cells in single files of endodermal cells (the responsible for these functions must be precisely regu- rate of cell formation being equal to the rate at which lated as the roots respond to the environment. These cells are lost from the meristem). The estimated time developmental changes are poorly understood. In the required for an individual cell to complete the formation case of salinity stress, the only organ that is directly of the strip after generation of the cell in the presence of exposed to excess salt is the root. Thus, it is important to 200 mM NaCl was not very different from that required understand the developmental and morphological in controls. Thus, salinity did not substantially affect the changes in the barrier structure that protects the roots actual process of formation of the strip in individual against an influx of elevated salt. Such an understanding cells. The radial width of the Casparian strip, a mor- should help select or generate salt-tolerant variants for phological parameter that should be related to the agricultural purposes. effectiveness of the strip as a barrier, increased in the The endodermis of virtually all vascular plants presence of 200 mM NaCl. The mean width of the lig- (Clarkson and Robards 1975; Haas and Carothers 1975) nified region was 0.92 lm in distilled water and 1.33 lm and the (Perumalla et al. 1990; Peterson and in 200 mM NaCl at the lowest position of the strip. The Perumalla 1990; Lehmann et al. 2000) of the roots of mean width of the strip relative to that of the radial wall many angiosperms develop a Casparian strip that is lo- at this position was significantly greater after growth in cated in the transverse and the radial walls of cells of the presence of 200 mM NaCl than in the controls, these tissues. When a root segment in which the Casp- namely, 20.5% in distilled water and 33.9% in 200 mM arian strip has already formed is immersed in hypotonic NaCl. These observations suggest that the function of solution, the plasmolysis figures of the endodermal cells the strip is enhanced under salt stress. appear to be stretched across the cells in cross-sections (Bryant 1934; Enstone and Peterson 1997). This phe- nomenon, known as band plasmolysis, indicates the tight adhesion of the plasma membrane to the wall at the Casparian strip. The where the strip forms is I. Karahara (&) Æ A. Ikeda Æ T. Kondo Æ Y. Uetake impregnated with hydrophobic materials, in particular Department of Biology, Faculty of Science, lignin and (Schreiber 1996; Zeier and Schreiber Toyama University, 930-8555 Toyama, Japan 1997; Zeier et al. 1999a, 1999b; Schreiber et al. 1999). E-mail: [email protected] Tel.: +81-76-4456630 Since the strip is considered to play a pivotal role as the Fax: +81-76-4456549 barrier to apoplastic transport in roots, it seems very 42 probable that the development and the morphology of include the modification of the cell wall with lignin and the strip should be regulated by environmental factors suberin and the tight adhesion of the plasma membrane (Karahara and Shibaoka 1994). to the cell wall. In the present study, the effect of salt The relationship between the development of the stress on the morphology of the strip was examined, and Casparian strip and the growth of roots was studied the relationship between anatomy and the function of initially in incense cedar (Wilcox 1962). It was subse- the strip as a barrier to the transport of solutes is quently demonstrated that the distance from the root tip discussed. to the lowest position of the exodermal strip decreased in maize roots under osmotic stress (Perumalla and Peter- son 1986) and in cotton roots under salt stress (Rein- Materials and methods hardt and Rost 1995). However, the mechanism responsible for Casparian strip development closer to Plant materials the root tip remains to be fully characterized. Kernels of maize (Zea mays L. ssp. sacchorata; Sakata Seed Co., Reinhardt and Rost (1995) concluded that salinity Yokohama, Japan) were soaked in distilled water for 24 h in accelerates the development of the Casparian strip from darkness at 25°C and then sown on moist vermiculite (Asahi Ko- their observation that the distance from the root tip to gyo Co., Okayama, Japan) that had been moistened with distilled the lowest position of the strip decreased under salt water (dH2O; control), or with a 100 mM or 200 mM solution of stress. However, this distance depends on the length and NaCl in dH2O (100 ml liquid per 250 ml of vermiculite). Using this brand of vermiculite, it has been confirmed that the root growth of number of cells in this region which, in turn, depends on the controls (grown in dH2O) was typical of roots with a supply of the rate of cell division in the meristem, the rate of cell 1 mM calcium sulfate (data not shown). Thus, calcium was not elongation, and the time required for formation of the added to the vermiculite. Seedlings were grown in darkness at 25°C strip in each individual cell. Therefore, one cannot for 4–8 days. The ages of seedlings are expressed in terms of the number of days from the start of imbibition. simply conclude that the development of the strip has accelerated exclusively from the observation that the strip is formed closer to the root tip. In the present Observations of the Casparian strip and band plasmolysis study, whether the development of the strip is actually by fluorescence and light microscopy accelerated in individual cells as a result of elevated salinity was ascertained. The lengths of primary roots that had grown for 8 days (counted from the start of imbibition) were measured and a frequency dis- Azaizeh and Steudle (1991) reported that salinity re- tribution was constructed. When the class width of the frequency duces the hydraulic conductivity of roots, and Azaizeh distribution was set at 50 mm, the most frequent length classes et al. (1992) proposed that the symplastic and transcel- were 150–199 mm in the control, 100–149 mm in 100 mM NaCl, lular pathway, or cell-to-cell path, might be responsible and 50–99 mm in 200 mM NaCl. The primary roots in the most for the decrease in the hydraulic conductivity. However, frequent length class for each treatment were used in all experi- ments. Hand-cut sections were prepared from the roots at intervals hydrostatic experiments with maize have shown that the of 1 mm for observations of the Casparian strip in the endodermis radial flow of water appears to be predominantly apo- and at intervals of 5 mm for observations of the strip in the exo- plastic (Frensch and Steudle 1989). We cannot exclude dermis, starting at the root tip. The term ‘‘root tip’’ is used to the possibility that the apoplastic path of water is also denote the apical end of the root in this study. Thus, the distance includes the root cap region. The sections were stained with 0.5% affected by NaCl (Azaizeh et al. 1992). One factor that (w/v) berberine hydrochloride (Nacalai tesque, Kyoto, Japan), might regulate hydraulic conductivity would be a change counterstained with 0.1% (w/v) aniline blue (Chroma Geselschaft, in the development and morphology of the Casparian Darmstadt, Germany; Brundrett et al. 1988) and then observed strip, which acts as a transport barrier. Differences in the under a fluorescence microscope as described previously (Karahara and Shibaoka 1998; Fig. 1). For fluorescence micrographs, 50-lm- hydraulic conductivity of maize roots caused by various thick frozen sections were prepared as described previously (Ka- growth conditions were shown to be due to differences in rahara and Shibaoka 1992). the development of the exodermal Casparian strip and Band plasmolysis was examined as follows. Segments (2 mm the suberin lamellae, and concomitant changes in levels long) of roots were cut from the region near the lowest position of of suberin and lignin were detected (Zimmermann et al. the Casparian strip, and immersed in phosphate-buffered saline (PBS; 10 mM NaH2PO4, 130 mM NaCl, pH 7.3) that contained 2000). Monitoring morphological changes in the Casp- 3.7% (v/v) formalin and 1 M urea for 1 h at room temperature. arian strip during its development, Barnabas and Pet- Then, 50-lm cross-sections were prepared as described above. erson (1992) found that the width of the endodermal Light photomicrographs were taken with a digital camera (HC- strip increased as its distance from the root tip increased 300Z; Fuji Photo Film Co, Tokyo, Japan) fitted to the microscope. in adventitious roots of onion. Moreover, natural radial widening of the strip occurs during the development of Quantification of cells in longitudinal sections the roots of vegetable marrow (Harrison-Murray and Clarkson 1973). However, the cited studies did not in- Segments (2 mm long) of roots were sampled from the mean clude any examination of roots under stress and it is location of the lowest position of the Casparian strip in controls clear that the morphology, for example the radial width, and in roots grown in 200 mM NaCl (see Table 1). Segments were of the strip itself should also be related to the effective- fixed for 12 h at 0°C in 50 mM sodium-cacodylate buffer (pH 7.2) that contained 2.5% glutaraldehyde. After washing in dH2O, seg- ness of the barrier (Lux and Luxova 2001). Major ments were post-fixed in 1% (w/v) osmium tetroxide in the same morphological characteristics of the Casparian strip buffer. The fixed segments were washed and dehydrated by passage 43 P -test was used to t =0 =0.0061 =0.5 =0.0050 =8.701 <0.0001 U t U Statistics -test or the U n ey Mean ± SE n Fig. 1a–c The Casparian strip in maize (Zea mays) primary roots, as observed under a fluorescence microscope. Frozen cross-sections were cut from 8-day-old roots that had been grown in the presence of 200 mM NaCl. a Section at a point 80 mm from the root tip. Exodermal Casparian strips were present but no endodermal strip

could be observed at this point because their staining was masked Mean ± SE by further modification of the wall. ex Exodermis, en endodermis. b Exodermal Casparian strip (arrowheads) at a point 55 mm from ) root tip on the number, length, and apparent rate of production of

the root tip. c Endodermal Casparian strip (arrowheads) at a point n 5 mm from the root tip. Bars = 100 lm(a), 20 lm(b,c) Zea mays through a graded ethanol series and embedded in Spurr’s resin (Spurr 1969). Longitudinal semi-thin 4-lm sections were cut such 169±762.7±2.1 5 1,694 – – – – 37.7±1.4 87±4 1029 6 that they included the center of the stele; sections were stained with DWMean ± SE 100 mM NaCl 200 mM NaCl Statistics/Probability an aqueous solution of 0.1% (w/v) sodium carbonate containing 0.5% (w/v) toluidine blue O. The cells in two files of endodermal cells were counted on a longitudinal section. Total numbers of cells in two files were averaged to give an estimate of cells per file. Since the part of the root that contained this region had been cut into segments, the number of cells did not equal the actual number in one complete cell file but was equal to the total number of cells in the segment. Since the apical organization of cells in maize root is of the closed type, it was possible to count the number of endo- dermal cells entirely as far as the initial cells.

Apparent rate of production of cells in an endodermal cell file

The time required for a cell to complete the formation of the strip was estimated by dividing the number of cells in an endodermal cell file in the region between the root tip and the lowest position of the strip by the number of cells produced in an hour in an endodermal cell file, namely, the apparent rate of production of endodermal cells in a cell file. Even though the rate of production of cells in a given file is not equal to the rate of cell division itself, it is pro- portional to the rate of cell division when this rate remains constant and it can be examined relatively easily in each file of cells. Moreover, in this study, it was necessary to know the rate of production of endodermal cells specifically and not the general rate of cell division. Since the growth rate of roots has been reported to increase for up to 3 days after the start of imbibition (Perumalla and Peterson 1986), the apparent rate of production of cells was examined using 4-day-old roots. Seedlings were grown in the presence or the ab- sence of 200 mM NaCl for 4 days. For determination of the m) apparent rate of production of cells, roots of the range within mean l ± 5 mm in length in the case of controls and of treatment with 200 mM NaCl were used (see Results). Roots were picked clean of Effects of salinity on the distance from the lowest position of the Casparian strip to the maize ( vermiculite and marked with indelible black ink (Magic Ink, Tokyo, Japan) under dim green light at 30 mm, in the case of and the root tip the root tip ( Length of primary rootsDistance examined from (mm) the lowestDistance position from of the the lowestNumber exodermal position of Casparian of cells strip the located to endodermal between the Casparian the root strip lowestLength tip to position of (mm) the of cells root the located tip endodermal between (mm) Casparian the strip lowest 84.8±4.0 position of 12.9±0.8 the endodermal Casparian strip and 8 8 71.9±4.9 6.1±0.7 8 8 34.9±4.0 3.2±0.2 10 10 – – 173.4±5.2 8 – – 127.1±3.7 8 78.2±5.2 10 – – Apparent rate of productionEstimated of time endodermal required cells for in formation one of cell the file endodermal (cells/h) Casparian strip in each cell (h) 19.3 – 8.7±0.8 – 6 – – 21.2 – 4.1±0.7 – 6 – – Table 1 controls, and at 14.5 mm, in the case of treatment with 200 mM endodermal cells located in thisdetermine region, whether and the on the effects estimated of time salinity required were forParameter formation statistically of significant the endodermal strip in individual cells. The Mann-Whitn 44

NaCl, from the root tip. The distances were 54% of the mean total salt stress, the numbers and the lengths of cells in length of the roots of 4-day-old seedlings from the root tip for each endodermal cell files in the region between the root tip treatment, namely, at a site at which elongation of cells had already ceased. The roots were returned to the vermiculite for 7.5 h. Then and the mean lowest position of endodermal Casparian roots were again removed from the vermiculite and marked at the strips were determined. Both the number and the length same distance from the root tip for each treatment. The distance of endodermal cells in this region decreased significantly between the two marks represented the elongation of roots during under salt stress (Table 1). the 7.5-h period. Segments (2 mm long) were cut from this region, If it is assumed that the rate of cell division remained fixed and embedded as described above. The endodermal cells in longitudinal sections were quantified as described above. The constant during the time when the cells in this region numbers of endodermal cells in two files observed in each section were produced, the numbers of cells produced in 7.5 h in were added together, divided by two and divided by 7.5 h. The an endodermal cell file should be equal to the number of result corresponded to the number of endodermal cells produced in cells produced per cell file at the root meristem in 7.5 h. 1 h in one cell file, namely, the apparent rate of production of cells in an endodermal cell file. The apparent rate of production of cells was examined using 4-day-old roots grown in the presence or the ab- sence of 200 mM NaCl. The roots of 4-day-old seedlings Quantitative analysis of the radial width of the Casparian strip were 55±3 mm (mean ± SE, n=67) long in the controls and 26±2 mm (n=73) long in 200 mM NaCl. The Ultrathin sections were cut from plasmolyzed segments prepared apparent rate of production of endodermal cells in one from the lowest position of the strip of roots grown either in dH2O cell file (Table 1) was reduced significantly under salt or in the presence of 200 mM NaCl. Segments (2 mm long) were sampled from the region 12–14 mm above the root tip in controls stress. If it is assumed that the apparent rate of pro- and 2–4 mm above the root tip in the case of treatment with duction of cells remained constant from 4 days after the 200 mM NaCl. The mean location of the lowest position of the start of imbibition, the estimated time required for an Casparian strip was located in this region. Each segment was im- individual cell to complete the formation of the strip mersed in a solution of 0.7 M mannitol in dH2O for 30 min, fixed and embedded as described above. Ultrathin cross-sections were under salt stress appears not very different from the time stained with aqueous uranyl acetate, with lead citrate and with 1% required in distilled water. KMnO4 for 1 min each. The lignified region of the strip was identified as the electron-dense region that was stained specifically with KMnO4 (Hayat 1986). The region of the cell wall to which the plasma membrane adhered tightly on plasmolysis and the radial Effects of salinity on the morphology of the Casparian width of the lignified region were measured for all Casparian strips strip in a given section. The radial width of the radial wall was defined as the width of the region in which the two radial walls of neighboring The width of plasmolyzed protoplasm in the radial endodermal cells appeared to be continuous. The experiment was direction was larger in the salt-stressed than in the repeated four times to confirm the reproducibility of the results. Since it was conceivable that the radial wall of endodermal cells control samples (Fig. 2). Major morphological charac- had also increased in width, the width of the radial wall was teristics of the Casparian strip were examined quanti- measured and the widths of the lignified region and the tightly tatively to determine whether the width of the lignified adhering region of the plasma membrane relative to that of the region and that of the tightly adhering region of the radial wall were calculated, as percentages. plasma membrane increased under salt stress. The widths of the lignified region and the tightly adhering region of the plasma membrane were significantly larger Results under salt stress than in the controls (Fig. 3, Table 2). The percentages were significantly larger under salt Effects of salinity on the distance from the root tip stress than in the controls (Table 2). to the lowest position of the Casparian strip

The distance from the root tip to the lowest position of Discussion the endodermal and the exodermal Casparian strip in roots of 8-day-old seedlings that had been grown in the Effects of salinity on the development and morphology presence or the absence of NaCl was examined of the Casparian strip at the cellular level (Table 1). The distance from the root tip to the lowest position of the strip decreased with increase in the In the primary roots of maize, the Casparian strip ma- concentration of NaCl in both the endodermis and the tured closer to the root tip under conditions of salt exodermis. stress. This result is consistent with results from cotton roots (Reinhardt and Rost 1995). It has been shown that formation of the strip at positions closer to the root tip Quantification of cells in endodermal cell files under salt stress was due to a decrease in the number of between the root tip and the lowest position cells and in the lengths of cells in the endodermis be- of the Casparian strip tween the root tip and the lowest position of the strip. The age of a cell at the lowest position of the strip is To ascertain why the distance from the root tip to the equivalent to the time required for the cell to complete lowest position of the Casparian strip decreased under the formation of the strip since the cell itself had been 45

Fig. 3a,b Effects of salinity on the ultrastructural morphology of the Casparian strip. Ultrathin cross-sections were prepared from plasmolyzed segments of maize roots. The radial walls between two Fig. 2a,b The endodermis in cross-sections prepared from plasmo- neighboring endodermal cells are shown and the electron-dense lyzed segments of maize roots grown under salt stress, as viewed portion of the wall, which was stained with KMnO4, corresponds under a light microscope. The plasmolysed protoplasm (arrow- to the strip. Brackets show the lignified region (L) and the tightly heads) appeared to be stretched across the cells. a Section at a point adhering region of the plasma membrane (P) of the Casparian 13 mm from the root tip in a control root. b Section at a point strip, and the radial wall (R). a Section at a point 13 mm from the 7 mm from the tip of a root grown in the presence of 200 mM root tip in a control root. b Section at a point 7 mm from the root NaCl. The plasmolyzed protoplasm appears to be wider in the tip in a root grown in the presence of 200 mM NaCl. The width of radial direction than that of the control. Bars = 10 lm the radial wall was measured along the wall, even though the brackets have been drawn as straight lines for convenience. Bars = 1 lm produced. It has been reported that the apparent rate of production of epidermal cells decreases under salt stress Therefore, the width was compared at the lowest posi- (Kurth et al. 1986; Zidan et al. 1990). In this study, the tion of the strip. estimated time that was required for a cell to complete formation of the strip under salt stress was not very different from the time in distilled water or was only Functional significance of changes in Casparian strip slightly longer. These observations indicate that salinity development and morphology did not have a major effect on the time-dependent for- mation of the strip itself in individual cells. The available evidence indicates that the root apical Previous reports that the endodermal Casparian strip zone is impermeable to ion movement (Lu¨ ttge and Weigl increases in radial width as it develops suggest that it is 1962; Enstone and Peterson 1992). On the other hand, in necessary to compare the width of the strip among cells the elongation zone of individual maize roots, namely, in which the strip itself is at the same developmental the region between the meristem and the lowest position stage. At present, the developmental stage of the strip of the endodermal strip, the mRNA that encodes a can be specified only at the initial stage of its formation. tonoplast aquaporin is expressed at high levels in the

Table 2 Effects of salinity on the width of the lignified region of the the width of the radial wall (as percentages) are also shown. The endodermal Casparian strip and the tightly adhering region of Mann-Whitney U-test was used to determine whether the effects of the plasma membrane. The widths of the lignified region and the salinity were statistically significant tightly adhering region of the plasma membrane relative to

Parameter Distilled water 200 mM NaCl Statistics/ Probability

Mean ± SE n Mean ± SE nU P

Width of the lignified region (lm) 0.92±0.03 26 1.33±0.07 22 77 <0.0001 Width of the tightly adhering region of the plasma membrane (lm) 0.85±0.02 50 1.16±0.05 44 386 <0.0001 Width of the lignified region relative to the width of the radial wall (%) 20.5±0.7 26 33.9±1.5 22 26 <0.0001 Width of the tightly adhering region of the plasma 18.7±0.4 50 29.5±1.0 44 51 <0.0001 membrane relative to the width of the radial wall (%) 46 endodermis and the pericycle (Barrieu et al. 1998). This Acknowledgements This work was supported by a Grant-in-Aid for suggests that water transport occurs in this region. The Scientific Research (no. 11740454) from the Ministry of Education, Science, Sports and Culture of Japan, by a Sasakawa Scientific decrease in the distance of this region may reduce loss of Research Grant from the Japan Science Society, and a grant for the water to the soil under salt stress. improvement of education from Toyama University to I.K. The The change in the width of the strip with the change authors are grateful to Professors M. Sugai, K. Masuda and in the environment raises an important possibility with S. Kamisaka of Toyama University for their helpful suggestions regard to the function of the strip. Initially, the radial and to Ms. H. Miyake for her skilled technical assistance. widening of the strip might serve to reinforce the func- tion of the strip as an apoplastic transport barrier. References However, for the reinforcement of the hydraulic resis- tance, at least the density of hydrophobic materials, such Azaizeh H, Steudle E (1991) Effects of salinity on water transport as lignin and suberin, must not decrease. This issue re- of excised maize (Zea mays L.) roots. Plant Physiol 97:1136– mains to be examined in future studies. However, if it is 1145 Azaizeh H, Gunse B, Steudle E (1992) Effects of NaCl and CaCl2 assumed that the widening of the strip results in rein- on water transport across root cells of maize (Zea mays L.) forcement of its hydraulic resistance, some useful seedlings. Plant Physiol 99:886–894 working hypotheses can be formulated, as follows. Baker DA (1971) Barriers to the radial diffusion of ions in maize If the Casparian strip itself actually allows limited roots. Planta 98:285–293 Barnabas AD, Peterson CA (1992) Development of Casparian passage of water and solutes, the radial width might be bands and suberin lamellae in the endodermis of onion roots. related to the effectiveness of the strip as a transport Can J Bot 70:2233–2237 barrier since the length of the resistant pathway would Barrieu F, Chaumont F, Chrispeels M (1998) High expression of be increased. The available evidence indicates that the the tonoplast aquaporin ZmTIP1 in epidermal and conducting tissues of maize. Plant Physiol 117:1153–1163 strip is impermeable to ion movement (Baker 1971; Brundrett MC, Enstone DE, Peterson CA (1988) A berberine- Nagahashi et al. 1974; Robards and Robb 1972; Leh- aniline blue fluorescent staining procedure for suberin, lignin, mann et al. 2000). On the other hand, there is no direct and callose in plant tissue. Protoplasma 146:133–142 evidence that the strip is a complete barrier to water Bryant AE (1934) A demonstration of the connection of the pro- transport. And there are reports that the apoplast is a toplasts of the endodermal cells with the Casparian strips in the roots of barley. New Phytol 33:231 major pathway for the radial transport of water under Clarkson DT, Robards AW (1975) The endodermis, its structural some conditions (Frensch and Steudle 1989; Peterson development and physiological role. In: Torrey JG, Clarkson et al. 1993), indicating that water can pass through the DT (eds) The development and function of roots. Academic strip. The radial width of the lignified region and the Press, London, pp 415–436 Enstone DE, Peterson CA (1992) The apoplastic permeability of tightly adhering region of the plasma membrane both root apices. Can J Bot 70:1502–1512 increased under salt stress. Those changes would hinder Enstone DE, Peterson CA (1997) Suberin deposition and band the passage of water and, thus, they would reinforce the plasmolysis in the maize (Zea mays L.) root exodermis. Can function of the strip as the barrier to apoplastic trans- J Bot 75:1188–1199 Frensch J, Steudle E (1989) Axial and radial hydraulic resistance to port. roots of maize (Zea mays L.) Plant Physiol 91:719–726 In addition to the morphological parameters of the Haas DL, Carothers ZB (1975) Some ultrastructural observations strip, the density in the strip and the composition of of endodermal cell development in Zea mays roots. Am J Bot lignin and suberin, the hydrophobic components of the 62:336–348 strip, should also be related to the effectiveness of the Harrison-Murray RS, Clarkson DT (1973) Relationships between structural development and the absorption of ions by the root strip as a barrier. These parameters should be analyzed system of Cucurbita pepo. Planta 114:1–16 in future studies by isolating strips (Karahara and Hayat MA (1986) Basic techniques for transmission electron Shibaoka 1992; Zeier et al. 1999a, 1999b) from roots. microscopy. Academic press, Orlando, FL A correlation between the width of the radial wall Karahara I, Shibaoka H (1992) Isolation of Casparian strips from pea roots. Plant Cell Physiol 33:555–561 and that of the strip has been observed even in intact Karahara I, Shibaoka H (1994) The Casparian strip in pea cells (Yokoyama and Karahara 2001). This suggests a epicotyls: effects of light on its development. Planta 192:269– pre-determined relationship between the radial growth 275 of wall and Casparian strip. However, in the present Karahara I, Shibaoka H (1998) Effects of Brefeldin A on the development of the Casparian strip in pea epicotyls. Protopl- study, any reproducible results indicative of the widen- asma 203:58–64 ing of the radial wall under salt stress were not obtained Kurth E, Cramer GR, Laeuchli A, Epstein E (1986) Effects of NaCl (data not shown). The width of the strip increased by a and CaCl2 on cell enlargement and cell production in cotton factor greater than would be expected from the increase roots. Plant Physiol 82:1102–1106 in length of the radial wall during growth (see Yokoy- Lehmann H, Stelzer R, Holzamer S, Kunz U, Gierth M (2000) Analytical electron microscopical investigations on the apo- ama and Karahara 2001); thus, some additional factor plastic pathways of lanthanum transport in barley roots. Planta must determine growth of the strip. Since salinity ap- 211:816–822 pears to be an environmental factor that regulates the Lu¨ ttge U, Weigl J (1962) Mikroautoradiographische Untersuch- 35 2) width of the Casparian strip, we plan to study the ungen der Aufnahme und des Transportes von SO4 und 45Ca2+ in Keimwurzeln von Zea mays und Pisum sativum L. mechanisms that control how and when the width of Planta 58:113–126 the strip is determined during the course of development Lux A, Luxova M (2001) Secondary dilatation growth in the root of endodermal cells. endodermis. In: Gasparikova O, Ciamporova M, Mistrik I, 47

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