Root border cells, which can be disengaged from root tips in water by mechanical stirring, come from meristematic tissue of root cap and consist of the water-solubility mucilage glue which is conjoint with root cap. Root border cells play an important role in regulating the root circumstance (Driouich et al., 2007; Wen et al., 2007). Release of Al-binding mucilage by border cells could play a role in protecting root tips from Al-induced cellular damages (Hawes et al., 2000). It has also been shown that border cells have different responses to microorganism infection (Vicré et al., 2005). At present, to research the growth control and biology functions of root border cells is one of the hotspots in this domain.

Iron toxicity is one of the main constraints to rice production in tropical and subtropical areas, where over 4 million hm² of land is adversely affected with yield reduction of approximately 30% to 60% (Cai et al., 2003; Majerus et al., 2007; Sahrawat, 2000). The direct iron toxicity that is harmful for the plant is Fe²⁺. When the plant assimilates superfluous Fe²⁺, it can cause illness. These years, root border cells involved in heavy metal tolerance have been largely studied in species such as pea (Pisum sativum) (Wen et al., 1999), snapbean (Phaseolus vulgaris) (Miyasaka and Hawes, 2001) and barley (Hordeum vulgare L.) (Zhu et al., 2003). The present investigation was undertaken to study the biotic characteristics and response to iron toxicity of root border cells in rice to search for iron tolerance mechanism at border cells.

Rice (Oryza sativa L.) seeds were surface sterilized with 0.1% (v/v) H₂O₂ solution, soaked in deionized water, and then germinated on wet filter paper in the dark at 30 °C until they sprouted. Then, the healthy and sterile seedlings were selected and transferred to 500 ml beakers, in which aeroponic culture with 200~250 ml dd H₂O was used to keep the surroundings humid (Zhu et al., 2003), and then the 500 ml beakers were put into the 1000 ml ones with 300 ml dd H₂O in the bottom to keep the surroundings warm (Cai et al., 2007). Nearly 80~100 seedlings were kept in each beaker.

To characterize the border cell number and viability, ten seedlings from eight categories (1, 2, 5, 10, 15, 20, 25 and 30 mm root length) were selected and the border cells from each of the seedlings were harvested into 1 μl FDA (fluorescein diacetate) and 2 μl PI (propidium iodide). After 10 min in the dark, the live cells showed green fluorescence and dead cells showed red fluorescence under fluorescent microscope (Motic BA400 EF-UPR, Motic Co., China). The ratio of live cells to all the cells counted was the activity of the root border cells.

Cells were harvested from 20~25 mm root length into water at approximately 24 border cells/µl. An equivalent volume of treatment solutions was added to result in a water control and 250 mg/L Fe²⁺ with 200 µmol/L CaCl₂ at pH 4.5. The cells were incubated in the dark at 25 °C, and cell viability was measured at 1, 4, 8, 12, 24 and 36 h after initiation of treatments. Five Fe²⁺ (FeSO₄·7H₂O) levels (0, 50, 150, 250 and 350 mg/L) were treated with 0.1 mmol/L Ca²⁺ at pH 4.5, and cell viability was measured at 4 h and 24 h after initiation of treatment.

Fig. 1 The dispersed border cells from root-tips of rice plant (FDA-PI staining, ×400). (a) The live border cells; (b) The dead border cells

Fig. 2 Number and viability of developed root border cells in rice

Fig. 3 Effect of Fe2+ exposure time on relative viability of border cells in vitro

Fe²⁺ treatment stimulated the death of detached border cells from rice cultivars (Fig.4). After 4 h Fe²⁺ treatment, the viabilities of 0 and 50 mg/L Fe²⁺ treatment were ≥80% and showed obvious discrepancy with the others (P<0.01). For the 150, 250 and 350 mg/L Fe²⁺ treatment, the viability of border cells decreased by 10% when the Fe²⁺ concentration increased by 100 mg/L. After 24 h Fe²⁺ treatment, the viabilities of all the Fe²⁺ levels were <65%. Border cells exhibited greater cell mortality for 24 h exposure to Fe²⁺ treatment compared with 4 h Fe²⁺ treatment. The viability of border cells decreased by 20% when the Fe²⁺ concentration increased by 100 mg/L. There was great harm to the viability of border cells suffered from iron toxicity. Border cells may be affected by microdosage Fe²⁺.

Fig. 4 Effect of Fe2+ concentration on viability of border cells in vitro

The effects of Fe²⁺ concentration and exposure time on border cells in vitro were observed. High Fe²⁺ concentration and 24 h Fe²⁺ exposure had inconspicuous effects on the viability of root border cells. However, with the increase of Fe²⁺ concentration and time, each factor showed significantly inhibition to viability of border cells. The decreased viability of border cells indicated that Fe²⁺ dosage and treatment time would cause deadly effect on the border cells. The increased cell death could protect the roots from toxin harm. Therefore, it may protect root from the damage caused by harmful iron toxicity.