AMINO ACID COMPOSITION, HEMICELLULOSE AND SOLUBLE SUGARS CONTENT IN NARROW-LEAVED LUPIN SEEDS (LUPINUS ANGUSTIFOLIUS L.) UNDER THE EFFECT OF REGLONE TURBO 200 SL

Barbara Adomas, Agnieszka I. Piotrowicz-Cieślak

ABSTRACT

The aim of the study was to determine the effect of Reglone Turbo 200 SL (diquat) as a desiccant applied to ‘Sonet’ narrow-leaved lupin on protein, amino acid, soluble carbohydrates and hemicellulose content in seeds. Reglone Turbo 200 SL was used at the dose of 2.0 lˇha^-1 on day 35 after lupin flowering. The diverse weather conditions significantly influenced the accumulation of some amino acids (threonine, proline, cysteine, glycine, tyrosine, leucine, histidine and lysine), content of soluble sugars and hemicellulose in the seeds tested. The desiccant applied to plants resulted in drought effect and hence an increase in soluble sugars content, particularly cyclitols (D-pinitol, D-chiro-inositol, myo-inositol). A similar effect was observed for hemicellulose. The desiccant applied did not modify the profile, content of amino acids and protein in the seeds tested.

Key words: amino acids, hemicellulose, lupin seeds, protein, Reglone Turbo 200 SL (diquat), soluble carbohydrates.

INTRODUCTION

Yellow lupin (Lupinus luteus L.), narrow-leaved lupin (Lupinus angustifolius L.) and white lupin (Lupinus albus L.) have all been developed as various cultivars adapted as agricultural crops for growth in the central part of Europe as well as in other parts of the word. In environmental protection, these plants may play a very important role in organic food production [17]. Narrow-leaved lupin (Lupinus angustifolus L.) is a valuable leguminous plant for medium firm soil and is recommended for growing in the Mazuria Province [22].

Lupin seeds are an important protein, vitamin and mineral source for animals. Narrow-leaved lupin seeds consist of 30 to 36% crude protein [19,18]. Lupin proteins are characterised by a low content of sulphur amino acids, especially methionine. The proteins of narrow-leaved lupin seeds have a higher content of threonine and tryptophan than the yellow lupin ones [9]. Also, the content of lysine, threonine and tryptophan in lupin seed proteins is lower than in the protein of soybean or other legumes [32]. Besides a desirable content of protein, the seeds have a high content of structural carbohydrates [10,6], which is undesirable for animals. Carbohydrates consist of the following two groups of compounds: easily assimilated inter-cellular carbohydrates and structural carbohydrates (cell wall components, for example, hemicellulose [3]).

Harvesting lupin seeds without loss while maintaining high quality is simple in principle but requires great skill and care, mainly because lupin seeds ripen unequally and stem and leaves dry slowly. Thus, it is sometimes useful to perform desiccation with Reglone 200 SL, especially on weeded plantations of yellow lupin, and with Harvade 250 SC (dimethypin) on non-weeded plantations [22]. Herbicides (e.g. diquat) are xenobiotics which are not essential compounds for plants growth and due to their high biological activity are known to cause stress in crops. As a result, they can cause changes in the content of some biochemically active endogenous substances in plants. According to Vlasenok et al. [29], herbicides modify the nutritional quality of seeds. Desiccants are applied when plants start to mature, thus accelerating and evening out the natural process. Chemical desiccation evokes in plants water stress which leads to many physiological, phenological and morphological changes [26]. Wate r stress is a basic parameter determining seed production [5].

The aim of the study was to determine the effect of Reglone Turbo 200 SL used as desiccant in ‘Sonet’ narrow-leaved lupin on protein, amino acids, hemicellulose and soluble carbohydrates contents in seeds.

MATERIAL AND METHODS

‘Sonet’ narrow-leaved lupin plants (Lupinus angustifolius L.) were grown on experimental plots near Olsztyn, Poland, over 1999-2000. The experimental plots were set up following the split-plot method, as randomized blocks, in three replications. Diquat was used (Reglone Turbo 200 SL) at the dose of 2.0 dm³·ha^-1 which equals 0.4 kg a.i.·ha^-1 on day 35 after plant flowering. The control plots were not treated with the desiccant.

The experimental plots were established in IV b class, lessive soil of good rye and potato complex consisting of loose silty sands and light clay pan. Winter triticale was used as the forecrop. The cultivation and fertilisation were carried out as recommended for lupin cultivation.

The analyses included the determination of crude nitrogen with the Kjeldahl method, protein amino acid composition with the use of an automated amino acid analyzer (AAA T 339 M) by Mikrotechna Praha after an adequate sample preparation according to Hirs et al. [12]. The tryptophan content was determined according to the Polish Standard PN-77, R-64820 [24] in a base hydrolysate. The contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined following van Soest and Winer [27], Southgate [28] method and the content of hemicellulose was calculated from the difference between ADF and NDF. The content of soluble sugars was determined following the method by Piotrowicz-Cieślak et al. [21]. Dry and fresh tissue matter was also examined. A total of 30 mg of tissue was homogenized in ethanol:water, 1:1 (v/v) containing 100µg phenyl-α-D-glucose as internal standard. Dry residues were derived with 300 µl of silylation mixture (trimethylsilylimidazole:pyrid yne, 1:1, v/v) in silylation vials at 70°C for 30 min. One µl of dried soluble carbohydrates was injected into a split-mode injector of a Shimadzu GC-14A gas chromatograph. Soluble carbohydrates were identified with intermediate standards as available and calculated from the ratios of area of peaks for each known carbohydrate to the area of peak for the internal standards.

The results were statistically analysed with variance analysis for two-factor experiments (split-plot).

RESULTS AND DISCUSSION

The weather conditions during both experimental years were distinct. The year 1999 was characterised by a rainfall deficiency and high temperatures, while the year 2000 had a high total rainfall and low temperatures during lupin development period (Fig. 1). According to Byrdy et al. [8], a good influence of desiccation after the application of Reglone Turbo 200 SL, is revealed particularly during rainy years, unfavourable to even plant ripening. In the study, high efficacy and quick effect of Reglone application was detected in rainy and cool year 2000.

The contents of protein, soluble carbohydrates and hemicellulose are presented in Table 1. The total protein content in the control lupin seeds averaged 34% of d.m. The Reglone desiccant applied did not affect the seed protein content. The average hemicellulose content was 3.6% in control seeds and significantly increased on average by 0.94 percentage point as an effect of Reglone Turbo 200 SL application in both years of investigations. The hemicellulose in lupin seeds is composed mostly of arabinogalactans and arabinans [2]. The hemicellulose accumulates during the first period in developing seeds, whereas the raffinose series of oligosaccharides accumulates later in seed development after cell wall deposition [7]. It can be hypothesised that hemicellulose accumulation depends on the weather conditions during initial stages of seeds development.

The temperature and Reglone Turbo 200 SL caused a decrease in the raffinose family oligosaccharides content (Table 2), particularly in stachyose. The contents of raffinose family oligosaccharides correlated with lupin seeds vigour and viability [11]. The plants responded to water deficit (e.g. after using Reglone Turbo 200 SL) by increasing the accumulation of polyols, and in particular their cyclic forms: myo-inositol, D-chiro-inositol and D-pinitol. The myo-inositol was dominant in the seeds. Accumulation of cyclitols and their methylated derivatives is correlated with desiccation. Cyclitols seem to function in two ways that are difficult to separate mechanistically: osmotic adjustment and osmoprotection [25]. Carbohydrates protect membrane phospholipids and proteins upon dehydratation. Hydroxyl groups of sugar may substitute water [30]. Koster [16] demonstrated that glass formation was dependent on a particular combination of sugar.

After application of Reglone Turbo 200 SL, the content of galactosyl cyclitols increased in seeds. Humidity and temperature during seed maturation are the factors which particularly modify the content of raffinose family oligosaccharides (RFO). The lupin seeds cultivated at the temperature of 15°C show a 30% greater level of RFO than the seeds cultivated at 28°C [20]. The sucrose and raffinose family oligosaccharides content in the seeds may indicate the storage capacity. The sucrose to RFO ratio reflects seeds storage capacity [14]. Seeds with the sucrose to RFO ratio value below 1 can be stored longer than 10 years. The greatest storage capacity was found under Reglone Turbo 200 SL in seeds harvested in 1999.

Different distinct temperature in the two years of the experiment had a significant effect on the amino acid profile, particularly proline (Table 3). The proline plays an important role as an osmoprotectant [13]. The content of methionine and lysine in control seeds protein in both analyzed vegetation seasons was similar and averaged 0.92g/16g N and 4.68 g/16g N for methionine and lysine, respectively. The control seeds protein contained low amounts of cystine and tryptophan, 1.48 and 0.88 g/16 g of protein, respectively. There is a necessity to increase biological value of lupin seeds protein (limited by deficiency of methionine, tryptophane and lysine) by addition of synthetic amino acids. The results obtained coincide with the results from previous lupin [1] and faba bean [23] experiments. The results confirmed a well-known principle [15] that methionine is the amino acid limiting the protein nutritional value. The desiccant applied did not affect the amino acid (AA ) profile. However, the AA profile was affected by the temperature variation.

Barrantes [4] has classified the amino acids into four categories: charged, hydrophilic, hydrophobic and apolar. The charged ones are basic and acidic. The basic amino acids are histidine, lysine and arginine. The acidic AA are aspartic acid and glutamic acid. The hydrophylic AA include all the charged ones plus threonine and serine. The hydrophobic group contains valine, methionine, isoleucine, leucine, tyrosine, phenylalanine and tryptophan. The last group of AA, apolar, includes hydrophobic AA minus tyrosine. The amino acid profiles in the narrow-leaved lupin seeds investigated appeared to be very similar. Glutamic acid was the most abundant amino acid in seeds, followed by aspartic acid with a frequency of four AA groups of ~33-34%. In the lupin seeds analyzed, the content of the total sum of amino acids was similar to that in soybean seeds [32]. The differences concerned the contents of each class of amino acids. In soybean seeds there are more apolar and hydrophobic AA and less acidic and the charged ones [31] than in the lupin seeds.

CONCLUSIONS

1. The weather conditions in the years 1999-2000 significantly modified the content of some amino acids (threonine, proline, cysteine, glycine, leucine, tyrosine, histidine and lysine) and the sum of soluble sugars and hemicellulose in narrow-leaved lupin seeds.

2. The influence of the desiccant applied on amino acid profile was non-significant.

3. Reglone Turbo 200 SL affected significantly the content of soluble sugars and hemicellulose in narrow-leaved lupin seeds.

1. Adomas B., Minakowski D., Murawa D., 2001. The protein and amino acids content in yellow lupin seeds under influence of desiccants. 4^th European Conf. on Grain Legumes, Cracow, 385.

2. Al-Kaisey M.T., Wilkie K.C.B., 1992. The polysaccharides of agricultural lupin seed. Carbohydrate Res. 227, 147-161.

3. Avigad G., Dey P.M., 1997. Carbohydrate metabolism: Storage carbohydrates. Plant Biochemistry, Academic Press Ltd., 143-204.

4. Barrantes F.J., 1975. The nicotinic cholineric receptor. Different composition evidenced by statistical analysis. Biochem. Biophys. Res. Commun. 54, 395-402.

5. Blackman S.A., Obendorf R.L., Leopold A.C., 1992. Maturation proteins and sugars in desiccation tolerance of developing soybean seeds. Plant Physiol. 100, 225-230.

7. Brenes A., Jansman A.J.M., Marquardt R.R., 2004. Recent progress on research on the effects of antinutritional factors in legume and oil seeds in monogastric animals. [In:] Recent advances of research in antinutritional factors in legume seeds and oilseeds. Proc. 4^th international workshop on antinutritional factors in legume seeds and oilseeds. Toledo (Spain), EAAP Publication 110, 195-208.

8. Brillouet J.M., Riochet D., 1983. Cell wall polysaccharides and lignin in cotyledons and hulls of seeds from various lupin (Lupinus L.) species. J. Sci. Food Agric. 34, 861-868.

9. Byrdy S., Górecki K., Łaszcz E., 1976. Pestycydy [Pesticides]. PWRiL [in Polish].

10. Eggum B.O., Beames R., Datta F.U., 1993. Protein and energy evaluation with rats of seed from 11 lupin cultivars. Anim. Feed Sci. Technol. 43 (1-2), 109-119.

11. Gdala J., Buraczewska L., 1996. Chemical composition and carbohydrate content of seeds from several lupin species. J. Anim. Food Sci. 5 (4), 403-416.

12. Górecki R.J., Piotrowicz-Cieślak A.I., Obendorf R.L., 1997. Soluble sugars and flatulence-producing oligosaccharides in maturing yellow lupin (Lupinus luteus L.) seeds. Seed Sci. Res. 7, 185-193.

13. Hirs C.H., Stein W., Moor H., 1954. The amino acid composition of ribonuclease. J. Biol. Chem. 211, 9411-9500.

14. Hong Z., Lakkineni K., Zhang Z., Verma D.P.S., 2000. Removal of feedback inhibition of ¹-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiol. 122, 1129-1136.

15. Horbowicz M., Obendorf R.L., 1994. Seed desiccation tolerance and storability: dependence on flatulence-producing oligosaccharides and cyclitols – review and survey. Seed Sci. Res. 4, 385-405.

16. Itenova K., Flengmark P., 1999. Seed quality of yellow lupin (Lupinus luteus L.) grown in Denmark. [In:] Lupin, an ancient crop for the new millennium. Proc. 9^th Inter. Lupin Conf., Klink-Műritz, Germany, 213.

17. Koster K.L., 1991. Glass formation and desiccation tolerance in seeds. Plant Physiol. 95, 302-304.

18. Longnecker N., Brennan R., Robson A., 1998. Lupin Nutrition. [In:] Lupins as crop plants: Biology, production and utilization. Eds. J.S. Gladstones, C.A., Atkins and J. Hamblin. CAB International, 121-148.

19. Martinez J.C., Escobedo M.R., Davila-Ortiz G., 2004. Effect of aqueous, acid and alkaline thermal treatments on the protein quality of Lupinus campestris seeds. [In:] Recent advances of research in antinutritional factors in legume seeds and oilseeds. Proc. 4^th international workshop on antinutritional factors in legume seeds and oilseeds. Toledo (Spain), EAAP Publication 110, 337-342.

20. Peterson D.S., Mackintosh J.B., 1994. The chemical composition and nutritive value of Australian grain legumes. Grains Research and Development Corporation, Canberra, Australia.

21. Piotrowicz-Cieślak A.I., 2002. Wpływ temperatury na zawartość cukrów rozpuszczalnych i jakość fizjologiczną nasion łubinu żółtego [Effect of temperature on the content of soluble saccharides and physiological quality of yellow lupin seeds]. Zesz. Probl. Post. Nauk Roln. 481, 205-210 [in Polish].

22. Piotrowicz-Cieślak A.I., Gracia Lopez P.M., Gulewicz K., 2003. Cyclitols, galactosyl cyclitols and raffinose family oligosaccharides in Mexican wild lupin seeds. Acta Soc. Bot. Pol. 72 (2), 109-114.

23. Piotrowicz-Cieślak A.I., Rutkowski M., Adomas B., 2000. Occurrence of cyclitols in developing lupin seeds tissues after application of Harvade 250SC. Horticulture and Vegetable Growing 19 (3), 1, 296-303.

24. Pisulewska E., Szymczyk B., Pisulewski P.M., 2001. Effect of plant desiccation Reglone on gross chemical composition, amino acid, and nutritive value of seed protein in determinate and indeterminate cultivars of faba beans (Vicia faba L. var. minor). 4^th European Conf. on Grain Legumes, Cracow, 384.

25. Polska Norma PN-77/R-64820 Pasze – Oznaczanie zawartości tryptofanu [Fodder – Tryptophan content determination] [in Polish].

26. Popp M., Lied W., Bierbaum U., Gross M., Grose-Schulte T., Hams S, Oldenettel J., Schuler S., Wiese J., 1997. Cyclitols – stable osmotica in trees. Trees, 257-270.

27. Sawhney V., Singh D.P., 2002. Effect of chemical desiccation at post-anthesis stage on some physiological and biochemical changes in the flag leaf of contrasting wheat genotypes. Field Crops Res. 77 (1), 1-6.

28. Soest van P., Winer R.H., 1967. Use of detergents in the analyses of fibrous feeds. IV. Determination of plant cell-wall constituents. J. Assoc. Off. Anal. Chem. 50, 50-55.

29. Southgate D.A., 1991. Determination of food carbohydrates. Elsevier Applied Sci. London, New York, 232.

30. Vlasenok L.J., Latypowa R.M., Tarasenko L.G., Khodasevich E.V., Shekunowa M.G., Yudaeva M.J., 1990. Effect of herbicides on photosynthetic apparatus in various lupin species. Fiziologiya Rast. 37 (2), 226-232.

31. Xiao L., Koster K.L., 2001. Desiccation tolerance of protoplasts isolated from pea embryos. J. Exp. Bot. 364, 2105-2114.

32. Zarkadas C.G., Voldeng H.D., Yu Ran Zi, Choi V.K., 1997. Determination of the protein quality of three new northern adapted cultivars of common and miso type soybeans by amino acid analysis. J. Agric. Food Chem. 45, 1161-1168.

33. Zarkadas C.G., Voldeng H.D., Yu Ran Zi, Choi V.K., 1999. Assessment of the protein quality of nine northern adapted yellow and brown seed coated soybean cultivars by amino acid analysis. J. Agric. Food Chem. 47, 5009-5018.

Agnieszka I. Piotrowicz-Cieślak
Department of Plant Physiology and Biotechnology
University of Warmia and Mazuria
Plac Łódzki 3, 10-720 Olsztyn, Poland

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed ‘Discussions’ in each series and hyperlinked to the article.