Suzuki et al. (1993) found a Thai variety Daw Dam lacking LOX-3, the major component of lipoxygenase isozymes in rice embryo, by monoclonal antibody technique. Compared with two rice varieties Koshihikari and Koganemochi having normal LOX-3 activity with optimum pH 7.0, Daw Dam showed a lower level of the peroxidation of unsaturated fatty acids (Suzuki et al. 1996a). Genetic studies also indicated that LOX-3 was controlled by a single gene, and the absence of LOX-3 was recessive (Suzuki et al. 1996b). Breeding rice varieties without LOX-3 is important for maintaining high quality during grain storage period (Suzuki et al. 2000).

To date, monoclonal antibody technique has been used to identify rice seeds lacking LOX-3. However, this method is expensive and time-consuming when screening a large segregating population. We reported here a rapid and inexpensive spectrophotometric method to screen LOX-3-null embryos based on the oxidative ability of 9-hydroperoxide, the catalytic products of LOX-3, by one color test based on the oxidation of iodide-starch (Hammond et al. 1992). This method was successfully combined with the specific inhibitor of lipoxygenase, nordihydroguaiaretic acid, when purified LOX-3 (Purified according to Ida et al. 1983 and Ohta et al. 1986) was exposed to emulsions containing linoleate (data not shown).

Two rice cultivars (Oryza sativa L.) were used in our experiments, Daw Dam and Koshihikari. Single embryo (50 embryos from each cultivar were used) was cut with a scalpel for immediate experiment, and incubated in a test tube containing 1 ml of solution A ( 0.08% Tween 20, borate buffer, 1mM linoleic acid, see details in Table 1) for 10 min at room temperature (25C) before adding 200 ml of solution B (5 ml saturated aqueous potassium iodide per 100 ml of 15% (v/v) acetic acid) and solution C (1% (w/v) soluble starch). Optical densities

of these solutions were measured at 410 nm (wave length of maximum absorbance peak of solution from Koshihikari showing orange color with faint purple) against distilled water as control.

All experiments were repeated at least five times. The results indicated that solution extracted from single embryo of Koshihikari with normal LOX-3 activity in pH 9.0 or 8.2 borate buffers and linoleic acid solution, which was further incubated with potassium iodide and starch, led to the formation of orange color with faint purple after 40 hr. In contrast, solution extracted form single Daw Dam embryo without LOX-3 activity displayed milky white with faint yellow. On the other hand, both of solutions extracted from single embryos of Koshihikari and Daw Dam showed milky white with purple by the 40hr treatment in pH 7.4 borate buffer, linoleic acid solution and further incubation with potassium iodide and starch.

As shown in Table 1, the sensitivity of spectrophotometric method, estimated by the difference in optimal density at 410 nm between Koshihikari and Daw Dam ( delta A₄₁₀= A_{410Koshihikari}- A_{410Daw Dam}), displayed a pH- and concentration-dependent response. Among borate buffers, 0.2, 0.4 mol/l at pH 9.0 and 0.4 mol/l at pH 8.2 buffers gave the significant differences in A₄₁₀ average between two cultivars with or without LOX-3 activity. These results coincided with the above evaluation due to color difference.

This method depends on the amounts of 9- or 13-hydroperoxides of linoleic acids produced by LOXs, which could oxidize the iodide ion to iodine, and the released iodine combined with starch develops a purple color with a high sensitive style. In this experiment, treatment in borate buffer at pH 9.0 or 8.2 can inhibit the activities of LOX-1 and LOX-2 showing optimum pH at 4.5 and 5.5, respectively. Therefore, the above treatment could diminish the interference of the LOX-1 and LOX-2 during the color developing process, and the sensitivity after 40 hr incubation stands for LOX-3 activity from Koshihikari. On the other hand, different concentrations of borate buffers could also change pH values in reaction system eventually, thus influencing the color development in this experiment. In this way, the treatment in 0.2 or 0.4 mol/l borate buffer at pH 9.0 was suitable in our experiment.

Using above screening method, we also found that some Japonica varieties (Wuyujing3, Nekken2, Nipponbare, Akihikari, Zhendao88 and Wunongzao) had relatively high LOX-3 activities in single embryo than those of some Indica varieties (Nanjing11, Zhen Zhu ai,Yuanfengzao, Teqing, Erjiuqing and Guichao2). This result may partly explain that Japonica varieties have lower storability compared with Indica varieties.

In conclusion, this new method do not require any special equipment, and is more rapid, inexpensive and convenient than monoclonal antibody technique.

References

Suzuki, Y., T. Nagamine, A. Kobayashi, K. Ohtsubo, 1993. Detection of a new rice variety lacking lipoxygenase-3 by monoclonal antibodies. Japan. J Breed. 43: 405-409

Suzuki, Y., T. Yasui, U. Matsukura, J. Terao, 1996a. Oxidative stability of bran lipids from rice [Oryza sativa (L.)] lacking lipoxygenase-3 in seeds. J. Agric. Food Chem. 44: 3479-3483

Suzuki, Y., T. Yasui, K. Okuno, 1996b. Genetic analysis of null-allele for lipoxygenase-3 in rice seeds. Euphytica. 91: 99-101

Suzuki, Y., K. Ise, T. Nagamine, 2000. Geographical variation of the gene, (lox3(t)), causing lipoxygenase-3 deficiency in Asian rice varieties. Rice Genet. Newsl. 17: 13-14

Hammond, E. G., D. N. Duvick, W. R. Fehr, D. F. Hildebrand, E.C. Lacefield, T. W. Pfeiffer, 1992. Rapid screening techniques for lipoxygenases in soybean seeds. Crop Sci. 32: 820-821

Ida, S., Y. Masaki, Y. Morita, 1983. The isolation of multiple forms and product specificity of rice lipoxygenase. Agric. Biol. Chem. 47: 637-641.

Ohta, H., S. Ida, B. Mikami, Y. Morita, 1986. Purification and characterization of rice lipoxygenase component 3 from embryos. Agric. Biol. Chem. 50: 3165-3171.