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Method to quantify root border cells in sandy soil
Soil Biology & Biochemistry 36 (2004) 1517–1519 www.elsevier.com/locate/soilbio
Short communication
Method to quantify root border cells in sandy soil Morio Iijimaa,*, Toshifumi Higuchia, Akira Watanabea, A. Glyn Bengoughb a
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan b Soil-Plant Dynamics Unit, Scottish Crop Research Institute, Dundee DD2 5DA, UK Received 8 January 2004; received in revised form 1 April 2004; accepted 18 April 2004
Abstract Root border cells are cells that detach from the growing root cap, and serve both physical and biological roles in the rhizosphere. Most work on border cells has been confined to agar, or hydroponic culture, because of the difficulty in separating them from soil particles. We present a new method to separate the root border cells from soil, and quantify border cell numbers in non-sterile sandy loam soil at contrasting matric potentials (220 and 2300 kPa). Recovery rates of 90 ^ 1% were achieved using a combination of surfactants, sonication, and centrifugation. Root border cell numbers in the dry soil (1.4 £ 103 after 24 h) were significantly decreased as compared with those in the wetter soil (1.7 £ 103 after 24 h). Possible reasons for the decreased release of border cells are discussed. q 2004 Elsevier Ltd. All rights reserved. Keywords: Maize; Matric potential; Mucilage; Root border cells; Root cap; Sloughed root cap cells
The root cap releases mucilage and sloughs thousands of intact living cells that decrease the soil frictional resistance to root growth (Iijima and Kono, 1992; Bengough and McKenzie, 1997; Iijima et al., 2000, 2003a,b). These root border cells have numerous biological roles in the rhizosphere (Hawes et al., 1998, 2000). The cells may help protect root tips from Al-induced cellular damage (Miyasaka and Hawes, 2001), nematode attack (Zhao et al., 2000), and pathogenic fungi (Hawes et al., 2000). Techniques are needed to quantify the numbers of border cells in soil, so that these roles can be investigated. This is a significant challenge because the cells may adhere to soil particles; to-date border cells have only been recovered from sand culture (Iijima et al., 2000), never soil. Iijima et al. (2000) separated root border cells from sand with a particle size of 125 –710 mm, and quantified cell release to the rhizosphere in loose and compacted sand. Cells were separated by the combination of dispersion in surfactant and sonication. Separation of border cells from soil is more difficult, however, due to the presence of clay and silt particles which may stick to and obscure the cells. The release of root border cells in soil is likely to be affected by the soil water status. In wet soil water they may release very easily whereas, in dry soil they may stick to * Corresponding author. Tel.: þ 81-52-789-4020; fax: þ81-52-789-5558. E-mail address: [email protected] (M. Iijima). 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.04.025
the root cap (Hawes and Brigham, 1992). In this paper, we described a technique to separate root border cells from soil particles, and then quantify the release of root border cells to the rhizosphere soil at two contrasting water contents.
1. Cell separation procedure Three surface-active agent, Tween 20, 80, and 85, and sodium pyrophosphate and hexa-metaphosphoric acid were tested for their efficacy in dispersing a mixture of cells and soil particles. A mixture of Tween 80 (100 mg g21) with sodium pyrophosphate (100 mg g21) was judged to be the most effective combination for cell separation. Maize (Zea mays L. cv. Robusto 30-71) seedlings were used for the experiment. The cell separation procedure was tested on separate border cell samples collected from individual maize roots. Maize seeds were surface-sterilised by immersion in a saturated solution of calcium hypochlorite for 5 min, and then washed several times with distilled water. They were placed on blotting paper moistened with distilled water in a Petri dish, and germinated at 28 8C for 48 h in the dark. The apical 20 mm of root was placed in an Ependorf tube filled with 1 ml of the mixture of Tween 80 with sodium pyrophosphate for 30 min at room temperature. Root border cells adhering loosely to the root cap surface
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M. Iijima et al. / Soil Biology & Biochemistry 36 (2004) 1517–1519
were separated from the root cap by sonication for 1 min. A 0.1 ml sub-sample was collected and placed on a glass slide. The initial cell number was counted at £ 100 magnification under a light microscope. An average of three subsample counts per root was used as the initial cell number (count 1; Fig. 1), and the entire separation procedure is summarised in Fig. 1. Next, 0.2 g of sandy loam soil (sand 56%, silt 36%, clay 8%; sieved , 2 mm) was added to the solution, agitated well, and left standing at room temperature for 30 min; the mass of soil added was approximately equivalent to the mass of rhizosphere soil around 20 mm of root. The solution was made up to 2 ml. The sucrose density gradient method was tried for separating cells from soil particles. However, it was ineffective due to border cells being dragged by soil particles into the higher density sucrose layer, decreasing recovery rates (results not presented). Instead, a combination of sedimentation in a centrifuge, together with sonication, was used to separate the border cells from soil. Average soil particle density was 2.61 g cm23, and the average density of root border cells was approximately 1.19 g cm23 (by the sucrose density gradient method). Settled sand particles (. 50 mm equivalent diameter according to Stokes Law) were removed 10 s after mixing the solution. Next, the supernatant of clay particles (, 2 mm diameter) were removed after 20 s centrifugation at 90g. This procedure was repeated three times to remove, theoretically, 98% of the clay particles according to the clay particle sedimentation rates. Brief sonication (about 5 s) followed by gentle shaking preceded each centrifugation. The cells were stained with aqueous Toluidine Blue O (30 mg g21). The cells were carefully counted after separation at £ 100 magnification as before (count 2; Fig. 1). We added
2700 ^ 100 cells and recovered 2400 ^ 100 cells recovered. Good recovery rates of 90 ^ 1% (mean ^ S.E. of six replicates plants) were obtained. The 10% loss of cells was mostly due to the soil particles remaining in solution interfering with cell counting under the microscope (soil particles between 2 and 50 mm remained). Counting the border cells under the microscope was the most timeconsuming part of the process, and was highly dependent on the quantity of silt present in the sample (e.g. could take 1 – 2 h per sample instead of 0.5 h per sample for solutiongrown roots). The silt content was the limiting factor in this technique, and we estimate that counting the cells may restrict the use of the technique in soils with greater silt contents (i.e. . 36%).
2. Border cell release as affected by soil water content Maize seedlings with straight seminal roots 18 ^ 1 mm long were transplanted into sandy loam soil packed to a dry bulk density of 1.15 g cm23 in an acrylic root box (125 £ 200 £ 6 mm). The soil was wetted to either 200 mg g 21 ( c ¼ 220 kPa; wet) or 80 mg g 21 ( c ¼ 2300 kPa; dry). Soil matric potential of bulk soil, not the rhizosphere soil, was measured using a thermocouple psychrometer and a tensiometer. Seedlings were grown for 24 h in darkness at 28 8C. Because the shoot does not emerge from the soil within 24 h growth period, the seedling was not exposed to the light. Seminal root length was measured for eight replicate plants in each treatment, and rhizosphere soil , 3 mm from the root axis was collected under the stereomicroscope. Soil particles and cells were separated using the method developed above, and border cell counts were compared between treatments using an Analysis of Variance in Microsoft Excel 2000. Root elongation did not differ significantly between treatments (Table 1). Root border cell numbers were decreased significantly in the dry treatment. The most likely explanation for this is that border cell release from the surface of the root cap was decreased in the dry treatment. It is also likely that the root border cells will adhere more strongly to the root cap in the drier soil, due to the more dehydrated state of the mucilage (Read et al., 1999). Table 1 Root elongation rates and numbers of root border cells collected from rhizosphere soil
Fig. 1. Technique used to separate the root border cells from soil particles in (a) cell recovery experiment; and (b) soil water content experiment.
Gravimetric water contents
Wet (20%)
Dry (8%)
Significant difference
Root elongation rate (mm d21) Root border cell number (1 £ 103)
25.6 ^ 1.35
25.5 ^ 1.50
NS
1.65 ^ 0.048
1.40 ^ 0.075
*
Values are mean ^ SE of eight replicates, * indicates significant difference between wet and dry treatment at P , 0:05:
M. Iijima et al. / Soil Biology & Biochemistry 36 (2004) 1517–1519
Vermeer and McCully (1982) first observed the border cells in soil, and stated that the cells survived for several days in rhizosphere soil. The quantification technique established can be applied to field soil to investigate the possible ecological roles of root border cells.
Acknowledgements Thanks to: Peter Barlow (Bristol University) and Shigenori Morita (Tokyo University), for helpful discussions; Japanese Society of Promotion of Science, Royal Society, and BBSRC for funding. The Scottish Office Agriculture, Environment and Fisheries Department provide grant-in-aid to SCRI.
References Bengough, A.G., McKenzie, B.M., 1997. Sloughing of root cap cells decreases the frictional resistance to maize (Zea mays L.) root growth. Journal of Experimental Botany 48, 885 –893. Hawes, M.C., Brigham, L.A., 1992. Impact of root border cells on microbial populations in the rhizosphere. Advances in Plant Pathology 8, 119 –148.
1519
Hawes, M.C., Brigham, L.A., Wen, F., Woo, H.H., Zhu, Y., 1998. Function of root border cells in plant health: pioneers in the rhizosphere. Annual Review of Phytopathology 36, 311 –327. Hawes, M.C., Gunawardena, U., Miyasaka, S., Zhao, X., 2000. The role of root border cells in plant defence. Trends in Plant Science 5, 128– 133. Iijima, M., Kono, Y., 1992. Development of Golgi apparatus in the root cap cells of maize (Zea mays L.) as affected by compacted soil. Annals of Botany 70, 207–212. Iijima, M., Griffith, B., Bengough, A.G., 2000. Sloughing of cap cells and carbon exudation from maize seedling roots in compacted sand. New Phytologist 145, 477 –482. Iijima, M., Barlow, P.W., Bengough, A.G., 2003a. Root cap structure and cell production rates of maize (Zea mays L.) roots in compacted sand. New Phytologist 160, 127–134. Iijima, M., Higuchi, T., Barlow, P.W., Bengough, A.G., 2003b. Root cap removal increases root penetration resistance in maize (Zea mays L.). Journal of Experimental Botany 54, 2105–2109. Miyasaka, S.C., Hawes, M.C., 2001. Possible role of root border cells in detection and avoidance of aluminum toxicity. Plant Physiology 125, 1978–1987. Read, D.B., Gregory, P.J., Bell, A.E., 1999. Physical properties of axenic maize root mucilage. Plant and Soil 211, 87–91. Vermeer, J., McCully, M.E., 1982. The rhizosphere in Zea: new insight into its structure and development. Planta 156, 45–61. Zhao, X., Schmitt, M., Hawes, M.C., 2000. Species-dependent effects of border cell and root tip exudates on nematode behavior. Nematology 90, 1238–1239.
Short communication
Method to quantify root border cells in sandy soil Morio Iijimaa,*, Toshifumi Higuchia, Akira Watanabea, A. Glyn Bengoughb a
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan b Soil-Plant Dynamics Unit, Scottish Crop Research Institute, Dundee DD2 5DA, UK Received 8 January 2004; received in revised form 1 April 2004; accepted 18 April 2004
Abstract Root border cells are cells that detach from the growing root cap, and serve both physical and biological roles in the rhizosphere. Most work on border cells has been confined to agar, or hydroponic culture, because of the difficulty in separating them from soil particles. We present a new method to separate the root border cells from soil, and quantify border cell numbers in non-sterile sandy loam soil at contrasting matric potentials (220 and 2300 kPa). Recovery rates of 90 ^ 1% were achieved using a combination of surfactants, sonication, and centrifugation. Root border cell numbers in the dry soil (1.4 £ 103 after 24 h) were significantly decreased as compared with those in the wetter soil (1.7 £ 103 after 24 h). Possible reasons for the decreased release of border cells are discussed. q 2004 Elsevier Ltd. All rights reserved. Keywords: Maize; Matric potential; Mucilage; Root border cells; Root cap; Sloughed root cap cells
The root cap releases mucilage and sloughs thousands of intact living cells that decrease the soil frictional resistance to root growth (Iijima and Kono, 1992; Bengough and McKenzie, 1997; Iijima et al., 2000, 2003a,b). These root border cells have numerous biological roles in the rhizosphere (Hawes et al., 1998, 2000). The cells may help protect root tips from Al-induced cellular damage (Miyasaka and Hawes, 2001), nematode attack (Zhao et al., 2000), and pathogenic fungi (Hawes et al., 2000). Techniques are needed to quantify the numbers of border cells in soil, so that these roles can be investigated. This is a significant challenge because the cells may adhere to soil particles; to-date border cells have only been recovered from sand culture (Iijima et al., 2000), never soil. Iijima et al. (2000) separated root border cells from sand with a particle size of 125 –710 mm, and quantified cell release to the rhizosphere in loose and compacted sand. Cells were separated by the combination of dispersion in surfactant and sonication. Separation of border cells from soil is more difficult, however, due to the presence of clay and silt particles which may stick to and obscure the cells. The release of root border cells in soil is likely to be affected by the soil water status. In wet soil water they may release very easily whereas, in dry soil they may stick to * Corresponding author. Tel.: þ 81-52-789-4020; fax: þ81-52-789-5558. E-mail address: [email protected] (M. Iijima). 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.04.025
the root cap (Hawes and Brigham, 1992). In this paper, we described a technique to separate root border cells from soil particles, and then quantify the release of root border cells to the rhizosphere soil at two contrasting water contents.
1. Cell separation procedure Three surface-active agent, Tween 20, 80, and 85, and sodium pyrophosphate and hexa-metaphosphoric acid were tested for their efficacy in dispersing a mixture of cells and soil particles. A mixture of Tween 80 (100 mg g21) with sodium pyrophosphate (100 mg g21) was judged to be the most effective combination for cell separation. Maize (Zea mays L. cv. Robusto 30-71) seedlings were used for the experiment. The cell separation procedure was tested on separate border cell samples collected from individual maize roots. Maize seeds were surface-sterilised by immersion in a saturated solution of calcium hypochlorite for 5 min, and then washed several times with distilled water. They were placed on blotting paper moistened with distilled water in a Petri dish, and germinated at 28 8C for 48 h in the dark. The apical 20 mm of root was placed in an Ependorf tube filled with 1 ml of the mixture of Tween 80 with sodium pyrophosphate for 30 min at room temperature. Root border cells adhering loosely to the root cap surface
1518
M. Iijima et al. / Soil Biology & Biochemistry 36 (2004) 1517–1519
were separated from the root cap by sonication for 1 min. A 0.1 ml sub-sample was collected and placed on a glass slide. The initial cell number was counted at £ 100 magnification under a light microscope. An average of three subsample counts per root was used as the initial cell number (count 1; Fig. 1), and the entire separation procedure is summarised in Fig. 1. Next, 0.2 g of sandy loam soil (sand 56%, silt 36%, clay 8%; sieved , 2 mm) was added to the solution, agitated well, and left standing at room temperature for 30 min; the mass of soil added was approximately equivalent to the mass of rhizosphere soil around 20 mm of root. The solution was made up to 2 ml. The sucrose density gradient method was tried for separating cells from soil particles. However, it was ineffective due to border cells being dragged by soil particles into the higher density sucrose layer, decreasing recovery rates (results not presented). Instead, a combination of sedimentation in a centrifuge, together with sonication, was used to separate the border cells from soil. Average soil particle density was 2.61 g cm23, and the average density of root border cells was approximately 1.19 g cm23 (by the sucrose density gradient method). Settled sand particles (. 50 mm equivalent diameter according to Stokes Law) were removed 10 s after mixing the solution. Next, the supernatant of clay particles (, 2 mm diameter) were removed after 20 s centrifugation at 90g. This procedure was repeated three times to remove, theoretically, 98% of the clay particles according to the clay particle sedimentation rates. Brief sonication (about 5 s) followed by gentle shaking preceded each centrifugation. The cells were stained with aqueous Toluidine Blue O (30 mg g21). The cells were carefully counted after separation at £ 100 magnification as before (count 2; Fig. 1). We added
2700 ^ 100 cells and recovered 2400 ^ 100 cells recovered. Good recovery rates of 90 ^ 1% (mean ^ S.E. of six replicates plants) were obtained. The 10% loss of cells was mostly due to the soil particles remaining in solution interfering with cell counting under the microscope (soil particles between 2 and 50 mm remained). Counting the border cells under the microscope was the most timeconsuming part of the process, and was highly dependent on the quantity of silt present in the sample (e.g. could take 1 – 2 h per sample instead of 0.5 h per sample for solutiongrown roots). The silt content was the limiting factor in this technique, and we estimate that counting the cells may restrict the use of the technique in soils with greater silt contents (i.e. . 36%).
2. Border cell release as affected by soil water content Maize seedlings with straight seminal roots 18 ^ 1 mm long were transplanted into sandy loam soil packed to a dry bulk density of 1.15 g cm23 in an acrylic root box (125 £ 200 £ 6 mm). The soil was wetted to either 200 mg g 21 ( c ¼ 220 kPa; wet) or 80 mg g 21 ( c ¼ 2300 kPa; dry). Soil matric potential of bulk soil, not the rhizosphere soil, was measured using a thermocouple psychrometer and a tensiometer. Seedlings were grown for 24 h in darkness at 28 8C. Because the shoot does not emerge from the soil within 24 h growth period, the seedling was not exposed to the light. Seminal root length was measured for eight replicate plants in each treatment, and rhizosphere soil , 3 mm from the root axis was collected under the stereomicroscope. Soil particles and cells were separated using the method developed above, and border cell counts were compared between treatments using an Analysis of Variance in Microsoft Excel 2000. Root elongation did not differ significantly between treatments (Table 1). Root border cell numbers were decreased significantly in the dry treatment. The most likely explanation for this is that border cell release from the surface of the root cap was decreased in the dry treatment. It is also likely that the root border cells will adhere more strongly to the root cap in the drier soil, due to the more dehydrated state of the mucilage (Read et al., 1999). Table 1 Root elongation rates and numbers of root border cells collected from rhizosphere soil
Fig. 1. Technique used to separate the root border cells from soil particles in (a) cell recovery experiment; and (b) soil water content experiment.
Gravimetric water contents
Wet (20%)
Dry (8%)
Significant difference
Root elongation rate (mm d21) Root border cell number (1 £ 103)
25.6 ^ 1.35
25.5 ^ 1.50
NS
1.65 ^ 0.048
1.40 ^ 0.075
*
Values are mean ^ SE of eight replicates, * indicates significant difference between wet and dry treatment at P , 0:05:
M. Iijima et al. / Soil Biology & Biochemistry 36 (2004) 1517–1519
Vermeer and McCully (1982) first observed the border cells in soil, and stated that the cells survived for several days in rhizosphere soil. The quantification technique established can be applied to field soil to investigate the possible ecological roles of root border cells.
Acknowledgements Thanks to: Peter Barlow (Bristol University) and Shigenori Morita (Tokyo University), for helpful discussions; Japanese Society of Promotion of Science, Royal Society, and BBSRC for funding. The Scottish Office Agriculture, Environment and Fisheries Department provide grant-in-aid to SCRI.
References Bengough, A.G., McKenzie, B.M., 1997. Sloughing of root cap cells decreases the frictional resistance to maize (Zea mays L.) root growth. Journal of Experimental Botany 48, 885 –893. Hawes, M.C., Brigham, L.A., 1992. Impact of root border cells on microbial populations in the rhizosphere. Advances in Plant Pathology 8, 119 –148.
1519
Hawes, M.C., Brigham, L.A., Wen, F., Woo, H.H., Zhu, Y., 1998. Function of root border cells in plant health: pioneers in the rhizosphere. Annual Review of Phytopathology 36, 311 –327. Hawes, M.C., Gunawardena, U., Miyasaka, S., Zhao, X., 2000. The role of root border cells in plant defence. Trends in Plant Science 5, 128– 133. Iijima, M., Kono, Y., 1992. Development of Golgi apparatus in the root cap cells of maize (Zea mays L.) as affected by compacted soil. Annals of Botany 70, 207–212. Iijima, M., Griffith, B., Bengough, A.G., 2000. Sloughing of cap cells and carbon exudation from maize seedling roots in compacted sand. New Phytologist 145, 477 –482. Iijima, M., Barlow, P.W., Bengough, A.G., 2003a. Root cap structure and cell production rates of maize (Zea mays L.) roots in compacted sand. New Phytologist 160, 127–134. Iijima, M., Higuchi, T., Barlow, P.W., Bengough, A.G., 2003b. Root cap removal increases root penetration resistance in maize (Zea mays L.). Journal of Experimental Botany 54, 2105–2109. Miyasaka, S.C., Hawes, M.C., 2001. Possible role of root border cells in detection and avoidance of aluminum toxicity. Plant Physiology 125, 1978–1987. Read, D.B., Gregory, P.J., Bell, A.E., 1999. Physical properties of axenic maize root mucilage. Plant and Soil 211, 87–91. Vermeer, J., McCully, M.E., 1982. The rhizosphere in Zea: new insight into its structure and development. Planta 156, 45–61. Zhao, X., Schmitt, M., Hawes, M.C., 2000. Species-dependent effects of border cell and root tip exudates on nematode behavior. Nematology 90, 1238–1239.