ABSTRACT

Monoclonal antibodies (mAbs) directed towards rapeseed protein type trypsin inhibitors (RTI) have been produced and used in studies of the inhibitor level in seeds and sprouts of oilseed rape. Seeds of Brassica napus L. cv. Jazz were germinated and samples were collected of seeds or sprouts for day 0 (imbibed seeds), 1, and 2 whereas germinated seeds from day 5, 7, and 9 were divided into roots, stems and green parts. The trypsin inhibitor levels of the different samples were measured by enzymatic assay and by ELISA based on mAbs toward RTI. Using the enzymatic assay a trypsin inhibitor level of 2 TIU/g dormant seed was found and this level was found to decrease per gram dry matter during germination. At 5 days after germination initiation the far predominant fraction of inhibitor activity was found in the green parts. These results were supported by the immunochemical analyses, which showed the same relative contents of inhibitor activity in the different samples.

INTRODUCTION

Protein type inhibitors with ability to inhibit proteinases, particularly serine proteinases, are widely distributed in plant seeds, including crucifers (Wilimowska-Pelc, 1985; Menegatti et al., 1992; Ceciliani et al., 1994). The physiological role of the inhibitors may be as storage proteins, regulators of endogenous proteinase activity or as defence against phytophagous insects and pathogenic microorganisms (Liener and Kakade, 1980).

The molecular properties of these inhibitors have led to a classification of the different inhibitors into families of which the Kunitz, Bowman-Birk and the potato inhibitor families are the main families. In cruciferous plants inhibitors have been described for Raphanus sativus L. (Ogawa et al., 1971), Brassica oleracea L. (Wilimowska-Pelc, 1985; Broadway, 1993), Brassica napus L. (Visentin et al., 1992; Ceciliani et al., 1994) and Sinapis alba L. (Menegatti et al., 1992) where inhibitors with sequence homology and similarities with respect to molecular weight and high pI values found in Brassica napus and Sinapis alba have been suggested to form a new family of inhibitors (Ceciliani et al., 1994).

Studies of inhibitors of the foliage of B. oleracea showed the presence of different inhibitors (Broadway, 1993). These inhibitors have molecular properties distinct from the inhibitors of other crucifers and from the seeds of B. oleracea (Wilimowska-Pelc, 1985), with lower pI values and high molecular weights. The B. oleracea foliage inhibitors were shown to elicit a possible effect on two phytopathogenic fungi specific for other plant species (grapes and pea) but had no effect towards a pathogen specific to cabbage (Lorito et al., 1994). Antifungal activity has also been found of 2S albumins of oilseed rape (Terras et al., 1994).

The nutritional aspects of inhibitors present in seeds used for food and feed have also been the subjects of various studies because these inhibitors are able to inhibit trypsin and chymotrypsin, both enzymes of the digestive tracts in animals. Hence too high levels of inhibitors have been suspected to be antinutritional (Liener and Kakade, 1980) and consequently various attempts are being made to decrease/lower the inhibitor contents of food and feed. However, in this connection the possible effects of the inhibitors for nutritional value (Eggum et al., 1995) and resistance of the plants should also be considered to exploit possible natural plant protection systems.

For leguminous plants, investigations have comprised evaluation of germination on inhibitor level reduction in relation to improved nutritional value (Frias et al., 1995) and investigation of the inhibitor profile (Sreerama and Gowda, 1998) or excretion level during germination as a possible defence mechanism of the plants (Tan-Wilson and Wilson, 1982). In the present work, the effect of germination on inhibitor levels in different seedling parts is investigated as an initiation of studies on the rape seed inhibitors’ biochemical function, effects in relation to nutritional value and as defence against pathogens to understand the potential value of the inhibitors for the plants.

MATERIALS AND METHODS

Germination

Rape seed (Brassica napus L., cv. Jazz) were soaked overnight and grown on wet filter paper in 2 different trays for use as two independent sets of material. Samples were withdrawn after 1, 2 5, 7 and 9 days and collected as whole seed or when possible divided into roots, stems and green parts, and the samples were then freeze dried.

Extraction

Samples (34-200 mg) were extracted in 2 ml ion exchanged water by homogenisation using UltraTurrax 10 N (Janke and Kunkel, Darmstadt, Germany) for 2 minutes followed by centrifugation at 1700 g for 10 minutes and pellets were discarded.

Trypsin inhibitor assay in microtiter trays

The trypsin inhibitor assays were performed in microtiter trays according to (Frøkiær et al., 1994) using N-α-benzoyl-L-arginine-4-nitroanilide (L-BAPA, Merck, Darmstadt) as substrate and an assay volume of 300 μl. Absorbance development was followed for 2 min. at 405 nm using an ELISA-reader (EL 340 from Bio-Tek Instruments). The trypsin inhibitor activity was expressed in trypsin inhibitor units (TIU) per g sample, where 1 trypsin inhibitor unit is the amount of inhibitor necessary for reduction of the trypsin activity by 1 unit under defined conditions. One unit is defined as the activity that catalyses transformation of 1 μmole L-BAPA into 4-nitroanilide per min. Trypsin inhibitor units were calculated in the range comprising 30-70 % inhibition.

Enzyme linked immunosorbent assay (ELISA)

Production and biotinylation of the monoclonal antibody is described in Frøkiær et al., 1994, which also describes the materials, conditions and buffers used for ELISA. In brief, purified RTI was immobilized to microtiter wells followed by removal of unbound RTI by washing. The wells were then incubated with a mixture of sample and biotinylated monoclonal antibody and unbound reagents were removed by washing. The final incubation comprised horseradish peroxidase conjugated streptavidine (P0397, Dako A/S, Glostrup, Denmark). Finally, the amount of monoclonal antibody bound to the immobilized phase was quantified as a function of the amount of tetramethylbenzidine converted. The absorbance was read at 450 nm using an ELISA reader (EL 340 from Bio-Tek Instruments).

RESULTS AND DISCUSSION

The level of trypsin inhibitor in rapeseed is for the investigated variety (B. napus var Jazz) found to be approximately 2 TIU/g seed. This is approximately the same level as found in the commonly used pea varieties with low inhibitor contents (Arentoft et al., 1994). Therefore, the same aspects of trypsin inhibitor influence on pancreas and protein utilization are relevant in relation to rapeseed inhibitors as discussed for legume inhibitors (Eggum et al., 1995).

Figure 1 shows the distribution on dry matter basis of the individual fractions obtained from the germinated seeds. For days 1 and 2, the seed could not be divided into different fractions. One day of germination was found to somewhat decrease (20-50%) the inhibitor level compared to ungerminated seeds. During the following days of germination, the trypsin inhibitor activity disappeared from the seed. Instead, inhibitor activity was detected in the green parts of the seedlings, whereas the stem and root fractions of the seedlings did not contain enzymatically detectable inhibitor activity. These results indicate that the protein inhibitors in the seed at the stage of germination are degraded similar to other storage proteins in the seed. Besides, the presence of the trypsin inhibitors in the leaves may indicate that the inhibitors are not merely storage proteins; the de novo synthesis of trypsin inhibitor in the leaves at the early stages of growth could perhaps be part of a plant protective system.

Figure 1. Seeds of Brassica napus cv. Jazz were germinated for up to nine days and for the last days divided into root, stem and green parts. The dry matter contents of each fraction are shown as bars relative to the total dry matter (RDM) in the fractions; whole seed (black), root (white), stem (hatched) and green parts (grey). The trypsin inhibitor contents of the fractions were determined enzymatically and are given as TIA per gram dry matter of examined material. TIA was found only for seeds at day 1 and 2 and for green parts at day 5, 7 and 9.

The ELISA method developed is specific for measurement of protein type trypsin inhibitors. The method is based on a competitive binding of antibody specific for RTI to either previously purified RTI immobilized to the surface of the microtiter well or to RTI of sample origin present in the incubation volume together with the antibody. Ungerminated seeds were chosen as internal standard included on each microtiter plate, and the levels of inhibitor in the individual samples were evaluated relative to this standard.

The transformation of data obtained by ELISA is illustrated in figure 2 together with the summarized ELISA results. The results found by ELISA follow the same tendency seen from the enzymatic measurements (figure 1), with a decrease in inhibitor content per gram dry matter and a disappearance of inhibitor from the seed and a formation of inhibitor in the stems and green parts. In addition, the ELISA method appears to have a lower detection limit than the enzymatic assay as low levels of inhibitor could be detected in the root and stem after five days of germination by ELISA.

Studies on electrophoretic patterns of inhibitors present in dormant seeds and after germination of horse gram (Dolichos biflorus) have shown that new inhibitor forms appear after germination (Sreerama and Gowda, 1998). These new forms appeared with the same electrophoretic properties as inhibitor forms found in leaf, flower and husk of flowering plants and at early stages of seed development (Sreerama and Gowda, 1998). It was not shown whether these forms were products of the same gene or not (Sreerama and Gowda, 1998). However, the inhibitor formation during seed maturation in horse gram seems to follow the routes of kidney bean, mung bean and adzuki bean, where products of the same genes are derived by post translational modifications (Sreerama and Gowda, 1998). These changes in inhibitor composition on germination may also be due to the

differences in molecular properties found for inhibitors of B. oleracea found by Wilimoska-Pelc, 1985, and Broadway, 1993.

Figure 2. Determination of RTI by use of a monoclonal antibody in ELISA. Left: illustration of the data obtained from competitive ELISA. Average absorption and standard deviation (n=6) of wells without inhibitor samples is illustrated by crosses. An extract of dormant seed (internal standard) prediluted 10 times are shown by squares. The dilution curve marked by diamonds is from an extract of undiluted stem fraction, day 5. The dotted line illustrates the level of 50% of maximum absorption, which is used for determination of sample dilution times for the individual samples. Right: Sample dilution ratios found to give 50% absorbance in ELISA was related to the dilution ratio found for dormant seed and given as relative values (RV). Two independent determinations was performed, illustrated by sets of white and black labels. Diamonds: dormant seed (day 1-2)/root (day 5-9); triangles: stems; circles: green parts.

CONCLUSION

Our studies have shown that the monoclonal antibody is able to recognize both the inhibitor present in the dormant seed and the inhibitors formed during germination. These results impair a great structural similarity between the inhibitors present at the two stages of developments hence indicating that the inhibitors may originate from the same genes. In addition, the use of antibodies for inhibitor determination will not be influenced by possible changes in levels of non-protein type trypsin inhibitors of e.g. phenolic origin. This is an important aspect both in relation to choice of methods for evaluation of the nutritive value of rapeseed protein and in relation to the action of trypsin inhibitors as plant protective agents.

ACKNOWLEDGEMENTS

S. Palmieri, ISCI, Bologna, Italy, is gratefully acknowledged for the kind donation of purified RTI for production of monoclonal antibodies. This work was financially supported by the Commission of European Union (Contract No. FAIR CT 95-0260).

REFERENCES

1. Arentoft, A.M., Frøkiær, H., Sørensen, H. and Sørensen, S. (1994) Determination of pea proteinase inhibitors using ELISA based on monoclonal antibodies. Acta Agric. Scand. Sect. B. Soil and Plant Sci. 44, 236-243.

2. Broadway, R.M. (1993) Purification and partial characterization of trypsin/chymotrypsin inhibitors from cabbage foliage. Phytochem., 33 (1), 21-27.

3. Broadway, R.M. and Missurelli, D.L. (1990) Regulatory mechanisms of tryptic inhibitor activity in cabbage plants. Phytochem., 29 (12), 3721-3725.

4. Ceciliani, F., Bortolotti, F., Menegatti, E., Ronchi, S., Ascenzi, P. and Palmieri, S. (1994) Purification, inhibitory properties, amino acid sequence and identification of the reactive site of a new serine proteinase inhibitor from oil-rape (Brassica napus) seed. FEBS Letters, 342, 221-224.

5. Eggum, B.O., Frøkiær, H., Mortensen, K., Olsen, L.R., Sørensen, H. and Sørensen, S. (1995) Physiological effects caused by isolated pea proteinase inhibitors included at different levels in a standard diet for rats. Proceedings of 2nd Eur. Conf. on Grain Legumes, 9-13 July, Copenhagen, DK, AEP (Eds), 270-271.

6. Frias, J., Diaz-Pollan, C., Hedley, C.L. and Vidal-Valverde, C. (1995). Evolution of trypsin inhibitor activity during germination of lentils. J. Agric. Food Chem., 43, 2231-2234

7. Frøkiær, H., Hørlyck, L., Barkholt, V., Sørensen, H. and Sørensen, S. (1994) Monoclonal antibodies against soybean and pea proteinase inhibitors: characterization and applications for immunoassays in food processing and plant breeding. Food Agric. Imm. 6, 63-72

8. Liener, I.E. and Kakade, M. (1980) Protease inhibitors, in Toxic Constituents of Plant Foodstuffs, 2nd edn. (Liener, I.E., ed.) Academic Press, New York, pp. 7-57.

9. Lorito, M., Broadway, R.M., Hayes, C.K., Woo, S.L., Noviello, C., Williams, D.L. and Harman G.E. (1994) Proteinase inhibitors from plants as a novel class of fungicides. Mol. Plant Micr. Interact. 7 (4), 525-527.

10. Menegatti. E., Tedeschi, G., Ronchi, S., Bortolotti, F., Ascenzi, P., Thomas, R.M., Bolognesi, M. and Palmieri, S. (1992) Purification, inhibitory properties and amino acid sequence of a new serine proteinase inhibitor from white mustard (Sinapis alba L.) seed. FEBS Letters, 301 (1), 10-14

11. Ogawa, T., Higasa, T. and Hata, T. (1971) Proteinase inhibitors in plant seeds. Part III. Characterization of two different types of inhibitors obtained from japanese radish seed (Raphanus sativus). Agric. Biol. Chem., 35 (59, 712-716.

12. Sreerama, Y.N. and Gowda, L.R. (1998) Bowman-Birk type proteinase inhibitor profiles of horse gram (Dolichos biflorus) during germination and seed development. J. Agric. Food Chem., 46, 2596-2600.

13. Terras, F.R.G., Schoofs, H.M.E., Thevissen, K., Osborn, R.W., Vanderleyden, J., Cammue, B.P.A. and Broekaert, W.F. (1993) Synergistic enhancement of the antifungal activity of wheat and barley thionins by radish and oilseed rape 2S albumins and by barley trypsin inhibitors. Plant Physiol. 103, 1311-1319.

14. Visentin, M., Iori, R., Valdicelli, L. and Palmieri, S. (1992) Trypsin inhibitory activity in some rapeseed genotypes. Phytochem., 31, 3677-3680.

15. Wilimowska-Pelc, A. (1985) Isolation and partial characterization of the trypsin inhibitor from the seeds of Brassica oleracea var. Sabellica. Acta Biochim. Polonica, 32 (4), 351-361.