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 roots 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 Lignin Æ Zea preventing the influx of salts into the stele through the apoplast 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 root 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 exodermis (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 cell wall 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 suberin (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.