ABSTRACT

Rapeseed meal is used as a protein supplement in animal feeds, especially after the development of low-erucic acid and low-glucosinolate cultivars (“canola”). However, the use of canola meal (CM) in certain feed formulations is limited due to a number of anti-nutritional factors present in the meal. Sinapine is one such factor. In this paper, we present the progress with respect to reducing the sinapine content of seeds in Brassica napus.

Sinapine, an antinutritional substance in canola meal

Rapeseed is grown principally for oil, and the global demand for canola-quality oil has increased the cultivation of canola varieties of Brassica napus and B. rapa. The meal component of the seed that remains after the oil has been extracted presents a good source of protein. We will refer to it as canola meal (CM) without making a distinction from its predecessor, rapeseed meal. CM contains about 38 – 40% of crude protein, and it is the second most abundant protein supplement in animal feed industry. However, the relatively poor digestibility of CM due to its fiber content, and the antinutritional factors such as phytate, sinapine and tannins detract from the full potential of CM (Bell, 1993). The concentration of phenolic compounds in Brassica spp. is at least 30 times higher when compared with soybean (Shahidi and Naczk, 1992). A substantial reduction of phenolics in CM would improve its use as a protein supplement in animals and perhaps move it toward a food-grade supplement (Kozlowska et al., 1990). We discuss the progress in reducing the sinapine content of B. napus by genetic engineering.

Sinapine is the most common of all phenolic esters in canola seeds. Phenolics and sinapine are derived from the general phenylpropanoid pathway (Strack et al., 1983; Chapple, 1994; Whetten et al., 1998; Fig. 1). Sinapine is a choline ester of sinapic acid, and constitutes 1-4 % of air-dried oil-free CM (Blair and Reichert, 1984; Uppstrom and Johansson, 1985). It imparts a bitter taste to canola meal and makes it less palatable to animals (Ismail et al., 1981). Further, its presence in the diet of certain strains of hens that lay brown-shelled eggs leads to an unacceptable fishy odour in the eggs (Pearson et al., 1980). Efforts to develop B. napus breeding lines with low sinapine content have not been successful. The lack of robust screening methods suitable for identifying any low-sinapine Brassica breeding lines and the amphidiploid nature of B. napus have made this task difficult. Sinapine is measured by chromatography (Clausen et al., 1983; Bouchereau et al., 1991) or colorimetry (Ismail and Eskin, 1979) of seed extracts. Recently, a nondestructive method that employs near infrared reflectance spectroscopy (NIRS) has been described (Velasco et al., 1998). Velasco and Mollers (1998) analyzed a collection of 1487 accessions from 21 Brassica species and 1361 samples of B. napus breeding lines for sinapic acid ester (SAE) content The lowest SAE content was at 1.7 g kg^-1 in B. tournefortii Gouan and at 5.0 g kg^-1 in a B. napus breeding line where as the average SAE content was at 8.0 g kg^-1. These results offer a possibility of reducing sinapine by plant breeding.

Fig.1. Schematic representation of sinapine biosynthesis in plants.

PAL : Phenylalanine ammonia lyase; C4H : Cinnamate 4-hydroxylase; C3H : Coumarate 3-hydroxylase; OMT : O-methyltransferase; FAH : Ferulic acid hydroxylase; SGT : sinapic acid: UDP-glucose sinapoyltransferase: SCT : sinapoylglucose:choline sinapoyltransferase. Biosynthesis of other phenolics from this pathway is not shown.

Metabolic target for manipulation of the sinapine content in Brassica napus

Sinapic acid is converted to sinapoyl glucose by sinapic acid:UDPG sinapoyltransferase (SGT) and then to sinapine by sinapoylglucose:choline sinapoyltransferase (SCT) (see Chapple et al., 1992; Fig.1). While the genes encoding SCT or SGT would be an obvious target for interdicting sinapine synthesis, they have neither been isolated nor their gene products well characterized. Therefore, we targeted an upstream step in the phenylpropanoid pathway. Chapple et al (1992) identified an A. thaliana mutant that had a defect in the conversion of ferulate to 5-hydroxyferulate and in the synthesis of sinpoyl esters. Subsequently, Meyer et al. (1996) isolated the A. thaliana gene for ferulic acid hydroxylase (FAH; f5h), and showed that FAH is a novel cytochrome P-450-linked monoxygenase that catalyzes the conversion of ferulic acid to 5-hydroxyferulic acid. Employing an f5h cDNA clone of Meyer et al. as a probe, we isolated its counterpart from a cDNA library of B. napus stem tissue.

Three f5h cDNA sequences that differed in restriction enzyme cleavage pattern and nucleotide sequence were further characterized. The f5h cDNA clones shared 81-83% identity in their nucleotide sequence. The deduced amino acid sequence was 93% identical to that of Arabidopsis f5h over the entire length (520 aa) of a predicted 58 kDa polypeptide.

Attempts to diminish ferulic acid hydroxylase (FAH) activity in Brassica napus as a means to reduce the sinapine content

A full-length f5h cDNA without the polyA end was inserted in antisense direction between a CaMV 35S promoter and a nos terminator (pJOY42) or between a napin promoter and a nos terminator in sense (pJOY43) or anti-sense direction (pJOY44). Plasmids pJOY42 and pJOY44 were expected to cause antisense suppression of f5h constitutively and in the mid-point of seed development, respectively, while pJOY43 was expected to cause overexpression of f5h in the seeds. The napin promoter (Kohno-Murase, et al., 1994) was chosen on the basis of the observation of Vogt et al. (1993), who found sinapine accumulation to occur at the midpoint in B. napus seed development. Agrobacterium tumefaciens-mediated transformation of B. napus cv. Westar hypocotyl explants was done (Moloney et al.,1989) and 22, 29 and 6 kanamycin resistant plants were regenerated from pJOY42-, pJOY43- and pJOY44-mediated transformation, respectively. Only those transgenic lines that had a single T-DNA insertion were studied further. It was important to use transgenic plants with single T-DNA insertion because segregation of multiple insertions in the seeds would not yield reliable information. The primary transgenic (T₀) plants with 35S-f5h gene construct (pJOY42) showed 40-60 % reduction in the sinapine content in comparison with that of the controls in HPLC analysis of methanol extracts (Table 1). The primary transgenic plants that showed lower levels of sinapine were selfed to yield homozygotes. The sinapine content in the fully mature seeds of the homozygous plants showed varying levels of sinapine reduction, with two of the transgenic plants (35S-A-1 & 4) showing a reduction of up to 40% (Table 1). The five transgenic plants transformed with pJOY42 appeared to be normal in growth and gross morphology in comparison with the parental line and plants transformed with vector that does not contain f5h.

Table 1. Relative sinapine content in the seeds of transgenic Brassica napus lines with f5h cDNA in sense/anti-sense orientation.

Note: The results are from HPLC analysis of methanol extracts of 10-seed samples from each plant. Four replicate analyses were performed. An authentic sinapine sample was used for quantitation. The asterick represents transgenic plant generated using pJOY44 gene construct (napin anti-f5h). nd, not determined.

In contrast to the transgenics constitutively expressing anti-f5h, transgenic plants containing sense or antisense f5h cDNA under the control of a napin promoter did not show any appreciable difference in the sinapine content (Table 1). This is surprising in view of the observation of Vogt et al, 1993 who found sinapine accumulation to start at about midpoint in seed development. Napin promoter is active at this phase of seed development (Kohno-Murase et al., 1994), and thus should have resulted in ample expression of the sense/antisense transcript. However, only one transgenic plant was analyzed in the case of pJOY44, hence we cannot conclude that the antisense expression of f5h transcript using napin promoter could not reduce sinapine content. On the other hand, we did not notice any increase in the sinapine content of plants transformed with pJOY43 plasmid construct. This suggests that the rate-limiting step in sinapine synthesis occurs further downstream of this step. Thus, while overexpression of f5h did not enhance sinapine production, curtailing it caused a reduction.

ACKNOWLEDGEMENTS

We are grateful to Clint Chapple (Purdue University) for providing us an Arabidopsis f5h cDNA clone. We thank Joe Hammerlindl for advice on genetic transformation, and Barry Panchuk and Don Schwab for nucleotide sequencing and oligonucleotide synthesis, respectively.

REFERENCES

1. Bell, J.M. (1993). Factors affecting the nutritional value of canola meal: A review. Can. J. Anim. Sci. 73:679-697.

2. Blair, R. and Reichert, R.D. (1984). Carbohydrate and phenolic constituents in a comprehensive range of rapeseed and canola fractions: nutritional significance for animals. J. Sci. food. Agric. 35:29-35.

3. Bouchereau, A., Hamelin, J., Lamour, I., Renard, M. and Larher, F. (1991). Distribution of sinapine and related compounds in seeds of Brassica and allied genera. Phytochemistry 30: 1873-1881.

4. Chapple, C. (1994). Genetic characterization of secondary metabolism in Arabidopsis. In Ellis B.E. (ed.) Genetic engineering of plant secondary metabolism. Plenum press, New York, 251-274.

5. Chapple, C., Vogt, T., Ellis, B.E. and Somerville, C.R. (1992). An Arabidopsis mutant defective in the general phenylpropanoid pathway. The Plant Cell 4:1413-1424.

6. Clausen, S., Olsen, O. and Sorensen, H. (1983). Separation of aromatic choline esters by high-performance liquid chromatography. J. chromatogr. 260:193-199.

7. Ismail, F. and Michael Eskin, N.A. (1979). A new quantitative procedure for determination of sinapine. J. Agric. Food. Chem. 27(4):917-918.

8. Ismail, F., Vaisey-Genser, M. and Fyfe, B. (1981). Bitterness and astringency of sinapine and its components. J. food Sci. 46:1241-1244.

9. Kohno-Murase, J., Murase, M, Ichikawa, H. and Imamura, J. (1994). Effects of an antisense napin gene on seed storage compounds in transgenic Brassica napus seeds. Plant Mol. Biol. 26:1115-1124.

10. Kozlowska, H., Naczk, M., Shahidi, F. and Zadernowski, R. (1990). Phenolic acids and tannins in rapeseed and canola. In. Shahidi (ed.) Canola and rapeseed. Production, chemistry, nutrition and processing technology. Van Nostrand Reinhold. New York, 193-210.

11. Meyer, K., Cusumano, J.C., Somerville, C. and Chapple, C.C.S. (1996). Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases. Proc. Natl. Acad. Sci. USA. 93:6869-6874.

12. Moloney, M.M, Walker, J.M. and Sharma, K.K. (1989). High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Reports 8:238-242.

13. Pearson, A.W., Butler, E.J. and Roger Fenwick, G. (1980). Rapeseed meal and egg taint: the role of sinapine. J. Sci. Food Agric. 31:898-904.

14. Shahidi, F. and Naczk, M. (1992). An overview of the phenolics of canola and rapeseed: chemical, sensory and nutritional significance. J. Am. Oil Chem. Soc. 69:917-924.

15. Strack, D., Knogge, W. and Dahlbender, B. (1983). Enzymatic synthesis of sinapine from 1-O-sinapoyl-β-D-glucose and choline by a cell-free system from developing seeds of red radhish (Raphanus sativus L. var. sativus). Z. Naturforsch. Teil. C. 38:21-27.

16. Uppstrom, B. and Johansson, M. (1985). Determination of sinapine in rapeseed. J. Swed. Seed Assoc. 95:123-128.

17. Velasco, L., Matthaus, B. and Mollers, C. (1998). Nondestructive assessment of sinapic

18. acid esters in Brassica species: I. Analysis by near infrared reflectance spectroscopy. Crop Sci. 38:1645-1650.

19. Velasco, L and Mollers, C. (1998). Nondestructive assessment of sinapic acid esters in Brassica species: II. Evaluation of germplasm and identification of phenotypes with reduced levels. Crop Sci. 38:1650-1654.

20. Vogt, T., Aebershold, R, and Ellis, B. (1993). Purification and characterization of sinapine synthase from seed of Brassica napus. Arch. Biochem. Biophys. 300 (2): 622-628.

21. Whetten, R.W., Mackay, J.J. and Sederoff, R.R. (1998). Recent advances in understanding lignin biosynthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:585-609.